Transcobalamin II Receptor Interacts with Megalin in the Renal Apical Brush Border Membrane

Purified human transcobalamin II receptor (TC II-R) binds to megalin, a 600 kDa endocytic receptor with an association constant, K(a), of 66 n M and bound(max) of 1.1 mole of TC II-R/mole of megalin both in the presence and absence of its ligand, transcobalamin II (TC II). Immunoprecipitation followed by immunoblotting of Triton X-100 extracts of the apical brush border membrane (BBM) from rabbit renal cortex revealed association of these two proteins. (35)[S]-TC II complexed with cobalamin (Cbl; Vitamin B(12)) bound to Sepharose-megalin affinity matrix and the binding was enhanced 5-fold when TC II-R was prebound to megalin. Megalin antiserum inhibited both the TC II-R-dependent and -independent binding of (35)[S]-TC II-Cbl to megalin, while TC II-R antiserum inhibited only the TC II-R-dependent binding. In rabbits with circulating antiserum to megalin, renal apical BBM megalin was present as an immune complex, but its levels were not altered. However, the protein levels of both TC II-R and the cation-independent mannose 6-phosphate receptor (CIMPR) were drastically reduced and the urinary excretion of TC II, albumin, and other low-molecular weight proteins was significantly increased. These results suggest that megalin contains a distinct single high-affinity binding site for TC II-R and their association in the native renal BBM is important for tubular reabsorption of many proteins, including TC II.     

Dietary Modifications of Phospholipid Composition and Biophysical Properties of the Brush Border Membrane Along the Trout Intestine

Brush border membranes (BBM) are isolated from middle and posterior intestine of trout fed either an essential fatty acid-rich diet or a saturated one. The different phospholipid classes are separated, and their fatty acid composition is determined. Fluorescence anisotropy studies are performed using two lipid fluorophores, namely diphenylhexatriene (DPH) and trimethyl-aminodiphenylhexatriene (TMA-DPH). The results indicate that the usual parameters affecting the lipid fluidity such as the phospholipid:protein (PL:PROT), cholesterol:phospholipid (CHOL:PL), and sphingomyelin:phosphatidylcholine (SP:PC) ratios and the unsaturation of the acyl chains are sufficient to explain the fluidity values determined using DPH, but not those obtained with TMA-DPH as a probe. This fluorophore is assessed to be localized only in the external leaflet of the membrane. Hence, it will be affected by the composition of the major phospholipids of this leaflet, sphingomyelin and phosphatidylcholine.     

Properties of Brush Border Vesicles Isolated from Rat Kidney Cortex by Calcium Precipitation

Brush border membrane vesicles were isolated from rat kidney cortex by differential centrifugation in the presence of 10 mM calcium. Their properties were compared to brush border vesicles isolated by free-flow electrophoresis. By the calcium precipitation method membrane vesicles were obtained in a shorter time with a similar enrichment of brush border marker enzymes (11- to 12-fold for alkaline phosphatase and maltase), with a similarly reduced activity of the marker enzyme for basal-lateral plasma membranes and an almost identical protein composition as revealed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The transport properties of the two membrane preparations for D-glucose, L-phenylalanine, and phosphate are essentially the same; there is some indication for a lower sodium permeability of the vesicles prepared by the calcium precipitation method. The latter vesicles were also shown to exhibit sodium gradient stimulated uptake of L-glutamate.     

Cholesterol Esterase Bound to Intestinal Brush Border Membranes Does Not Accelerate Incorporation of Micellar Cholesterol into Absorptive Cells

We confirmed that cholesterol esterase accelerated the incorporation of unesterified cholesterol solubilized in bile salt micelles into differentiated Caco-2 cells under various experimental conditions. Rat pancreatic juice and bovine cholesterol esterase increased the incorporation of micellar cholesterol into rat intestinal brush border membranes. The incorporation of micellar cholesterol was not changed in the brush border membranes enriched in and depleted of cholesterol esterase. The results suggest that the accelerated incorporation of micellar cholesterol by cholesterol esterase into absorptive cells is not mediated by the enzyme bound to the brush border membranes.     

Galectin-2 at the enterocyte brush border of the small intestine

The brush border of pig small intestine is a local hotspot for β-galactoside-recognizing lectins, as evidenced by its prominent labeling with fluorescent lectin PNA. Previously, galectins 3-4, intelectin, and lectin-like anti-glycosyl antibodies have been localized to this important body boundary. Together with the membrane glycolipids these lectins form stable lipid raft microdomains that also harbour several of the major digestive microvillar enzymes. In the present work, we identified a lactose-sensitive 14-kDa protein enriched in a microvillar detergent resistant fraction as galectin-2. Its release from closed, right-side-out microvillar membrane vesicles shows that at least some of the galectin-2 resides at the lumenal surface of the brush border, indicating that it plays a role in the organization/stabilization of the lipid raft domains. Galectin-2 was released more effectively from the membrane by lactose than was galectin-4, and surprisingly, it was also released by the noncanonical disaccharides sucrose and maltose. Furthermore, unlike galectin-4, galectin-2 was preferentially coimmunoisolated with sucrase-isomaltase rather than with aminopeptidase N. Together, these results show that the galectins are not simply redundant proteins competing for the same ligands but rather act in concert to ensure an optimal cross-linking of membrane glycolipids and glycoproteins. In this way, they offer a maximal protection of the brush border against exposure to bile, pancreatic enzymes and pathogens.     

Galectin-4 in normal tissues and cancer

Galectin-4 belongs to a subfamily of galectins composed of two carbohydrate recognition domains within the same peptide chain. The two domains have all the conserved galectin signature amino acids, but their overall sequences are only approximately 40% identical. Both domains bind lactose with a similar affinity as other galectins, but their respective preferences for other disaccharides, and larger saccharides, are distinctly different. Thus galectin-4 has a property of a natural cross-linker, but in a modified sense since each domain prefers a different subset of ligands. Similarly to other galectins, galectin-4 is synthesized as a cytosolic protein, but can be externalized. During development and in adult normal tissues, galectin-4 is expressed only in the alimentary tract, from the tongue to the large intestine. It is often found in relatively insoluble complexes, as a component of either adherens junctions or lipid rafts in the microvillus membrane, and it has been proposed to stabilize these structures. Strong expression of galectin-4 can be induced, however, in cancers from other tissues including breast and liver. Within a collection of human epithelial cancer cell lines, galectin-4 is overexpressed and soluble in those forming highly differentiated polarized monolayers, but absent in less differentiated ones. In cultured cells, intracellular galectin-4 may promote resistance to nutrient starvation, whereas—as an extracellular protein—it can mediate cell adhesion. Because of its distinct induction in breast and other cancers, it may be a valuable diagnostic marker and target for the development of inhibitory carbohydrate-based drugs. Published in 2004. This revised version was published online in July 2006 with corrections to the Cover Date.     

Water Permeability of Brush Border Membrane Vesicles from Kidney Proximal Tubule

Brush border membrane vesicles (BBMV) maintain an initial hydrostatic pressure difference between the intra- and extravesicular medium, which causes membrane strain and surface area expansion (Soveral, Macey & Moura, 1997). This has not been taken into account in prior osmotic water permeability P _f evaluations. In this paper, we find further evidence for the pressure in the variation of stopped-flow light scattering traces with different vesicle preparations. Response to osmotic shock is used to estimate water permeability in BBMV prepared with buffers of different osmolarities (18 and 85 mosM). Data analysis includes the dissipation of both osmotic and hydrostatic pressure gradients. P _f values were of the order of 4 × 10⁻³ cm sec⁻¹ independent of the osmolarity of the preparation buffer. Arrhenius plots of P _f vs. 1/T were linear, showing a single activation energy of 4.6 kcal mol⁻¹. The initial osmotic response which is significantly retarded is correlated with the period of elevated hydrostatic pressure. We interpret this as an inhibition of P _f caused by membrane strain and suggest how this inhibition may play a role in cell volume regulation in the proximal tubule. Received: 8 August 1996/Revised: 4 March 1997     

A Creatine Transporter Is Operative at the Brush Border Level of the Rat Jejunal Enterocyte

Although ergogenic effects and health benefits have been reported for creatine used as nutritional supplement, to date little is known about the mechanism of creatine absorption in the small intestine. Thus the current study was undertaken to elucidate the mechanism of creatine intake in rat jejunum with the use of well-purified brush border membrane vesicles, isolated from jejunal enterocyte. Creatine uptake was found markedly stimulated by inwardly directed Na⁺ and Cl^– gradients, potential-sensitive, strongly reduced by the substitution of Na⁺ and Cl^– with various cations and anions and positively affected by intravesicular K⁺. Moreover, creatine uptake is: 1) significantly inhibited by creatine stuctural analogs, 2) abolished by low concentrations of 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA), 3) saturable as a function of creatine concentration with an apparent Michaelis-Menten constant of 24.08±0.80 M and a maximal velocity of 391.30±6.19 pmoles mg protein^–1 30 s^–1. The transport is electrogenic since at least two Na⁺ and one Cl^– are required to transport one creatine molecule. Western blot analysis showed the same amount of creatine transport protein in the jejunal apical membrane when compared to ileum. Thus, these data demonstrate the existence of a Na⁺- and Cl^–-dependent, membrane potential-sensitive, electrogenic carrier-mediated mechanism for creatine absorption in rat jejunal apical membrane vesicles, which is biochemically and pharmacologically similar to those observed in other tissues. However, in other cell types the stimulatory effect of intravesicular K⁺ was never detected.     

铜离子胁迫下玉米根边缘细胞数量及存活率

采用悬空培养及离体培养的方式,观察玉米的根边缘细胞在铜离子胁迫下发生的形态、数量及存活率变化情况,结果表明,在玉米根长为32mm的时候,边缘细胞的数量达到最大,为4125个,但其存活率此时最低。玉米的根边缘细胞随着铜离子浓度增加,边缘细胞的数量发生变化。当处理浓度为50umol·L-1时,边缘细胞的死细胞数量达到758个（存活率为24．80％）,随后铜离子处理浓度增加到100umol·L-1时,存活率降低了17．54％。玉米根边缘细胞的数量随着铜离子浓度的增加呈现出逐渐减少的趋势,而存活率逐渐降低,说明在重金属铜离子胁迫下,根边缘细胞的释放起到对根际区域的保护作用。     

高等植物根边缘细胞的发育调控及其生物学功能

植物根边缘细胞是从根冠表皮游离出来并聚集在根尖周围的一群特殊细胞,以前曾称为根冠脱落细胞.最近的证据表明,绝大多数物种边缘细胞具有生物学活性,其发育是受内外信号调控.边缘细胞一旦从根表皮游离后,其代谢活性大大上升、基因表达明显不同于根冠细胞.最近,与边缘细胞发育早期和晚期相关的两个基因PsUGT1和RCPME1分别被克隆和鉴定.边缘细胞能特异性地合成、分泌一系列的化学物质,包括花色素苷、抗生素、特异性酶类及其他化学物质能抑制或促进根际周围的细菌、真菌、病毒、线虫等的生长以及中和根际周围一些有毒化学物质如铝毒.因此,边缘细胞在植物生长发育过程中起着多种生物学功能.     

高等植物根边缘细胞的发育调控及其生物学功能

植物根边缘细胞是从根冠表皮游离出来并聚集在根尖周围的一群特殊细胞 ,以前曾称为根冠脱落细胞。最近的证据表明 ,绝大多数物种边缘细胞具有生物学活性 ,其发育是受内外信号调控。边缘细胞一旦从根表皮游离后 ,其代谢活性大大上升、基因表达明显不同于根冠细胞。最近 ,与边缘细胞发育早期和晚期相关的两个基因PsUGT1和RCPME1分别被克隆和鉴定。边缘细胞能特异性地合成、分泌一系列的化学物质 ,包括花色素苷、抗生素、特异性酶类及其他化学物质能抑制或促进根际周围的细菌、真菌、病毒、线虫等的生长以及中和根际周围一些有毒化学物质如铝毒。因此 ,边缘细胞在植物生长发育过程中起着多种生物学功能     

A Thiamine/H+ Antiport Mechanism for Thiamine Entry into Brush Border Membrane Vesicles from Rat Small Intestine

Outwardly oriented H⁺ gradients greatly enhanced thiamine transport rate in brush border membrane vesicles from duodenal and jejunal mucosa of adult Wistar rats. At a gradient pH_in5:pH_out7.5, thiamine uptake showed an overshoot, which at 15 sec was three times as large as the uptake observed in the absence of the gradient. Under the same conditions, the binding component of uptake accounted for only 10–13% of intravesicular transport. At the same gradient, the K _m and J _max values of the saturable component of the thiamine uptake curve after a 6 sec incubation time were 6.2 ± 1.4 μm and 14.9 ± 3 pmol · mg⁻¹ protein · 6 sec⁻¹ respectively. These values were about 3 and 5 times higher, respectively, than those recorded in the absence of H⁺ gradient. The saturable component of the thiamine antiport had a stoichiometric thiamine: H⁺ ratio of 1:1 and was inhibited by thiamine analogues, guanidine, guanidine derivatives, inhibitors of the guanidine/H⁺ antiport, and imipramine. Conversely, the guanidine/H⁺ antiport was inhibited by unlabeled thiamine and thiamine analogues; omeprazole caused an approximately fourfold increase in thiamine transport rate. In the absence of H⁺ gradient, changes in transmembrane electrical potential did not affect thiamine uptake. At equilibrium, the percentage membrane-bound thiamine taken up was positively correlated with the pH of the incubation medium, and increased from about 10% at pH 5 to 99% at pH 9. Received: 17 July 1997/Revised: 16 September 1997     

小鼠肝癌及肝正常组织B4GalT糖基转移酶家族mRNA表达差异及其对细胞膜相关糖链的影响

探讨肝癌模型鼠与正常小鼠肝组织B4GalT(β-1,4-半乳糖转移酶)家族mRNA表达差异以及对细胞膜相关糖链的影响.采用RT-PCR方法检测肝癌模型鼠和正常对照小鼠肝癌组织中B4GalT家族7个成员以及唾液酸α-2,3转移酶ST3GalⅢ、ST3GalⅣ、ST3GalⅥ、α-1,6-岩藻糖转移酶FUT8 mRNA表达差异,应用凝集素芯片检测细胞膜表面半乳糖、岩藻糖、唾液酸表达情况.结果显示:与正常对照组相比,肝癌模型鼠肝组织中B4GalT-1和B4GalT-3、ST3GalⅣ和ST3GalⅥ、FUT8呈现高表达,肝癌细胞膜半乳糖、岩藻糖、唾液酸类型糖链增加,提示B4GalT-1和B4GalT-3与肝癌细胞膜半乳糖链增加相关.由于细胞Galβ-1,4-GlcNAc糖表位在ST3GalⅢ、ST3GalⅣ或ST3GalⅥ催化下与唾液酸α-2,3连接生成s-lewis x抗原前体,本实验中B4GalT-1和B4GalT-3与ST3GalⅣ、ST3GalⅤ、FUT8 mRNA表达具有相关性,提示B4GalT-1和B4GalT-3可能与ST3GalⅣ、ST3GalⅥ以及FUT4协同作用,导致肝癌细胞膜半乳糖、岩藻糖、唾液酸类型糖链增加.     

N-linked glycan-dependent interaction of CD63 with CXCR4 at the Golgi apparatus induces downregulation of CXCR4

Efficient downregulation of CXCR4 cell surface expression by introduction of the CD63 gene has previously been reported by us. In the present study, it was found that CD63 and its mutant efficiently interact with CXCR4 in live cells and that CD63-induced downregulation and interaction are significantly abrogated by the N- linked glycosylation inhibitor, TM. Furthermore, the downregulation and interaction were clearly attenuated by alternation of all three N- linked glycosylation sites in CD63. Either CD63 or CD63ΔN formed a complex with CXCR4 at the Golgi apparatus and the late endosomes, while CD63 GD mutants lost the ability to form a complex with CXCR4 exclusively at the Golgi apparatus. These findings suggest that CD63 interacts with CXCR4 through the N- linked glycans-portion of the CD63 protein and that the complex induces direction of CXCR4 trafficking to the endosomes/lysosomes, rather than to the plasma membrane. At the Golgi apparatus, there may be lysosome protein (CD63)-associated machinery that influences trafficking of other membrane proteins.     

白藜芦醇通过降低galectin-3 的表达抑制宫颈癌SiHa细胞的增殖并促进其凋亡

目的：探讨白藜芦醇对人宫颈癌SiHa 细胞增殖和凋亡的影响及galectin-3 在其中的作用。方法：以体外培养的人宫颈癌 SiHa 细胞为研究对象,实验分为对照组、白藜芦醇处理组及[白藜芦醇+galectin-3(5、10、20 ug/mL)]共5 组,分别给予生理盐水、 300 umol/L白藜芦醇及[300 umol/L白藜芦醇+Galectin-3 (5、10、20 ug/mL)]处理。48 小时后,分别收集各组细胞,采用MTT法检 测细胞的增殖情况,Caspase-3 活性试剂盒测定细胞的凋亡情况,Western Blot技术测定细胞内galectin-3 的蛋白表达水平。结果： 300 umol/L的白藜芦醇明显抑制SiHa 细胞的增殖,并显著增加其凋亡水平,同时减少了细胞内galectin-3 的蛋白表达。在白藜芦 醇处理的同时,给予5、10 及20 ug/mL 的Galectin-3,随着Galectin-3 蛋白浓度的增加,SiHa 细胞的增殖抑制率逐渐降低,凋亡水 平也逐渐下降,但是VEGF-R3 受体的磷酸化水平却逐渐升高。结论：白藜芦醇通过降低galectin-3 的表达抑制宫颈癌SiHa 细胞 的增殖并促进其凋亡。     

白藜芦醇通过降低galectin-3的表达抑制宫颈癌SiHa细胞的增殖并促进其凋亡

目的：探讨白藜芦醇对人宫颈癌SiHa细胞增殖和凋亡的影响及galectin-3在其中的作用。方法：以体外培养的人宫颈癌SiHa细胞为研究对象,实验分为对照组、白藜芦醇处理组及[白藜芦醇＋galectin-3（5、10、20μg/mL）]共5组,分别给予生理盐水、300μmol/L白藜芦醇及[300μmol/L白藜芦醇＋Galectin-3（5、10、20μg/mL）]处理。48小时后,分别收集各组细胞,采用MTT法检测细胞的增殖情况,Caspase-3活性试剂盒测定细胞的凋亡情况,Western Blot技术测定细胞内galectin-3的蛋白表达水平。结果：300μmol/L的白藜芦醇明显抑制SiHa细胞的增殖,并显著增加其凋亡水平,同时减少了细胞内galectin-3的蛋白表达。在白藜芦醇处理的同时,给予5、10及20μg/mL的Galectin-3,随着Galectin-3蛋白浓度的增加,SiHa细胞的增殖抑制率逐渐降低,凋亡水平也逐渐下降,但是VEGF-R3受体的磷酸化水平却逐渐升高。结论：白藜芦醇通过降低galectin-3的表达抑制宫颈癌SiHa细胞的增殖并促进其凋亡。     

Galactosyltransferase activity is not localized to the brush border membrane of human small intestine

A recent report [Rothet al. (1985)J. Cell Biol. 100: 118–125], using immunocytochemical techniques, calimed that human duodenal galactosyltransferase is located predominantly on the external aspect of enterocyte brush border membranes. Analytical subcellular fractionation by sucrose density gradient centrifugation of human jejunum biopsy homogenates demonstrated that galactosyltransferase activity is localized to the Golgi fraction (equilibrium density of 1.14 g cm^–3) and is not found in significant amounts in the brush border membrane (equilibrium density of 1.22 g cm^–3).     

植物细胞质膜离子通道研究进展

多种有机和无机离子作为重要的营养物质、渗透物质、辅酶和信号分子, 参与植物生殖、生长发育和逆境反应等多种生物学过程。离子通道是离子跨质膜和内膜运动的重要渠道和动态调控因子, 直接影响和调控细胞内离子浓度及亚细胞分布的动态变化。目前, 植物尤其是模式植物拟南芥(Arabidopsis thaliana)的多个离子通道家族被先后鉴定出来, 其中部分离子通道蛋白定位在细胞质膜上, 其基本生物学功能, 诸如蛋白结构、离子选择性和通透性、门控特点、活性调控机理以及不同离子通道之间的协同关系等均取得重要进展。该文概要介绍近年来植物细胞质膜离子通道方面的研究进展。     

Effects of ochratoxin A on membrane phospholipids of the intestine of broiler chickens,practical consequences

Ochratoxin A (OTA) is a mycotoxin produced by various species of Aspergillus and Penicillium. Ochratoxin A was classified as a group 2B carcinogen and is one of the major intestinal pathogenic mycotoxins. One of the most frequent modes of intoxication is consumption of contaminated food with mycotoxins. Feed represents the major cost and has a direct impact on the economical viability of broiler’s production system, since it must contain the necessary elements that allow the animal to express the maximum genetic potential while providing its nutritional requirements. Thus, the animal has to digest the feed and absorb its nutrients, which is in direct correlation with the gastrointestinal tract, especially the small intestine and the development of the mucosal surface area. Once ingested, OTA is absorbed by passive diffusion, mainly the jejunum. Ochratoxin A’s presence affects lipid membranes and could lead to the degradation of their normal structure and functionality. All of these effects contribute to the development of malabsorption. It was very interesting to study the effect of OTA on the layer of phospholipids of the bowel. The experimental group received OTA (0.05 to mg/kg BW) through an intra-peritoneal injection, every other day for 21 days. We noted that feed conversion ratio and average daily gain were reduced. Histological studies showed important alterations at the level of the mucosal membrane of the intestine (villosities, crypts) following intra-peritoneal administration of the mycotoxin. Thinning and enlargement at the base of the villosities, hyperplasia and crypts in irregular forms, blunting and denudation were observed through the examination of intestinal morphology. Biochemical studies, such as total lipid and phospholipid compositions, allowed us to have more detailed results. All identified mucosal phospholipids were modified, particularly the phosphatidylcholine (PC) and the phosphatidylethanolamine (PE) in the jejunum mucosa. In fact, there was a decrease by 55.81% for PC, 56.66% for PE, while a significant increase by 32.91% was noted for phosphatidylserine in the jejunum. It was very interesting to study the effect of OTA on the phospholipids layer of the bowel, as the mucous membrane of the small intestine represents the main site of absorption and transformation of nutriments. To avoid such disturbances and prevent the effects of the OTA, precautions must be taken to inhibit mold growth at the level of the feed manufactory units. Phosphatidylcholine and PE administrations may represent an option that could allow reestablishment of phospholipid equilibrium in the intestine.     

Ion Channels Induced in Planar Lipid Bilayers by the Bacillus thuringiensis Toxin Cry1Aa in the Presence of Gypsy Moth (Lymantria dispar) Brush Border Membrane

The apical brush border membrane, the main target site of Bacillus thuringiensis toxins, was isolated from gypsy moth (Lymantria dispar) larval midguts and fused to artificial planar lipid bilayer membranes. Under asymmetrical N-methyl-d-glucamine-HCl conditions (450 mm cis/150 mm trans, pH 9.0), which significantly reduce endogenous channel activity, trypsin-activated Cry1Aa, a B. thuringiensis insecticidal protein active against the gypsy moth in vivo, induced a large increase in bilayer membrane conductance at much lower concentrations (1.1–2.15 nm) than in receptor-free bilayer membranes. At least 5 main single-channel transitions with conductances ranging from 85 to 420 pS were resolved. These Cry1Aa channels share similar ionic selectivity with P _Cl/P _NMDG permeability ratios ranging from 4 to 8. They show no evidence of current rectification. Analysis of the macroscopic current flowing through the composite bilayer suggested voltage-dependence of several channels. In comparison, the conductance of the pores formed by 100–500 nm Cry1Aa in receptor-free bilayer membranes was significantly smaller (about 8-fold) and their P _Cl/P _NMDG permeability ratios were also reduced (2- to 4-fold). This study provides a detailed demonstration that the target insect midgut brush border membrane material promotes considerably pore formation by a B. thuringiensis Cry toxin and that this interaction results in altered channel properties. Received: 23 February 2001/Revised: 15 June 2001