Search

Introduction ATP binding cassette (ABC) membrane transporter multidrug resistance 1 (MDR1) is one of the most important efflux transporters in the human placenta protecting the fetus from exposure to xenobiotic toxicity. Recent studies have focused on placental MDR1 expression, but few studies have analyzed placental MDR1 transport activity. The purpose of this study was to investigate placental MDR1 transport activity using a relatively large sample size of human placentas. Furthermore, the effect of ABCB1 gene polymorphisms were investigated along with physiological factors including maternal age, times of pregnancy, BMI, delivery mode or pregnancy complications on placental MDR1 transport activity. Methods A total of 252 human placentas were obtained after delivery. MDR1 transport activity was detected by N-methyl quinidine uptake in placental microvillus membrane vesicles (MVMVs). Nine common single nucleotide polymorphisms (SNPs) in ABCB1 genes were determined by Snapshot. The association between ABCB1 gene polymorphisms, maternal age, times of pregnancy, BMI, delivery mode or pregnancy complications, and transporter activity was investigated. Results Inter-individual variations of MDR1 transport activity were observed among 252 subjects. The per unit protein activity was ranged from 0.05 to 0.15/mg. Nine SNPs in ABCB1 gene didn't exhibit significant association with transporter activity of MDR1. Likewise, neither age, times of pregnancy, delivery mode nor pregnancy complications showed any significant effect of placental MDR1 transport activity. But placental MDR1 transport activity in obese pregnant women was lower than those in non-obese pregnant women. Conclusion Inter-individual variations of MDR1 transport activity existed in human placentas. This may contribute to variations in drug exposure to the fetus affecting clinical outcomes. Maternal age, times of pregnancy, delivery mode nor pregnancy complications included in this study maybe not significantly impact placental MDR1 transport activity, but maternal obese could inhibit placental MDR1 activity.

Excessive iron increases the incidence of diabetes and worsens diabetic complications. Reciprocally, diabetes induces iron loading, partially attributable to elevated intestinal iron export according to a recent report. Herein, we show that iron uptake and the mRNA expression of iron importer divalent metal transporter 1 (DMT1) were significantly increased in the duodenum of streptozotocin-induced diabetic mice. Immunofluorescence staining of human intestinal biopsies revealed increased brush border membrane (BBM) and decreased cytoplasmic DMT1 expression in patients with diabetes, suggesting translocation of DMT1. This pattern of DMT1 regulation was corroborated by immunoblotting results in diabetic mice showing that BBM DMT1 expression was increased by 210%, in contrast to a 60% increase in total DMT1. PKC mediates many diabetic complications, and PKCα activity was increased in diabetic mouse intestine. Intriguingly, diabetic mice with PKCα deficiency did not show increases in iron uptake and BBM DMT1 expression. High-glucose treatment increased plasma membrane DMT1 expression via the activation of PKCα in cultured IECs. Inhibition of PKCα potentiated the ubiquitination and degradation of DMT1 protein. We further showed that high glucose suppressed membrane DMT1 internalization. These findings demonstrate that PKCα promotes microvillus membrane DMT1 expression and intestinal iron uptake, contributing to diabetic iron loading.—Zhao, L., Bartnikas, T., Chu, X., Klein, J., Yun, C., Srinivasan, S., He, P. Hyperglycemia promotes microvillus membrane expression of DMT1 in intestinal epithelial cells in a PKCα-dependent manner.

Objective Ileal transposition (IT) procedure leads to higher secretion of incretin hormones what is associated with a beneficial metabolic effect. However, IT will also have an influence on the related jejunum and ileum function. The aim of this research was to investigate the morphology of the jejunum and transposed ileum with the use of light and transmission electron microscopy (TEM) in order to determine the local alternations in the intestine resulting from the transposition. Methods Twenty male, 8-week-old, obese Zucker rats underwent IT and six of them sham surgery. To compare both groups, the transection was made at all corresponding ileum positions among both groups of animals. The ileal anastomoses among the rats of sham procedure were subsequently formed accordingly without IT. Three months following the surgery, the tissue samples of jejunum and ileum were harvested. Results A significant increase in villus length, a decrease in the crypt depth, and an increased thickness of mucosa-muscularis-serosa (MMS) as well as cellular hyperplasia, with increased mitochondrial density of the transposed ileum segment, were observed among the group of rats which underwent IT comparing to the ones undergoing sham surgery. In rats undergoing IT, microvillus degeneration in jejunum regions was observed. Conclusions Ileal transposition alters the morphology and ultrastructure of the ileum as well as the jejunum. Given that the microvillus membrane represents an important aspect of the enterocyte functions, a further biochemical and molecular research is necessary in order to assess whether the observed changes are beneficial or not and to explore the phenomenon of gut adaptability after metabolic surgery.

Chitosan is a polycationic polysaccharide consisting of β-(1-4)-linked glucosamine units and due to its mucoadhesive properties, chemical derivatives of chitosan are potential candidates as enhancers for transmucosal drug delivery. Recently, glycol chitosan (GC), a soluble derivative of chitosan, was shown to bind specifically to lipid raft domains in model bilayers. The small intestinal brush border membrane has a unique lipid raft composition with high amounts of glycolipids cross-linked by lectins, and the aim of the present work therefore was to study the interaction of FITC-conjugated GC (FITC-GC) with the small intestinal epithelium. Using organ culture of pig jejunal mucosal explants as a model system, we observed widespread binding of luminal FITC-GC to the brush border. Only little uptake via constitutive endocytosis into apical early endosomes occurred, unless endocytosis was induced by the simultaneous presence of cholera toxin B subunit (CTB). Biochemically, GC bound to microvillus membrane vesicles and caused a change in the density profile of detergent resistant membranes (DRMs). Collectively, the results showed that FITC-GC binds passively to lipid raft domains in the brush border, i.e. without inducing endocytosis like CTB. Instead, and unlike CTB, FITC-GC seems to exert a stabilizing, detergent-protective effect on the lipid raft organization of the brush border.

From fits of drug transport kinetics across confluent MDCKII-hMDR1-NKI and Caco-2 cell monolayers we estimated the levels of efflux active P-glycoprotein (P-gp) in these two cell lines (companion paper). In the present work, we compared the efflux active P-gp number to the total P-gp level, using liquid chromatography-tandem mass spectrometry, and showed that in Caco-2 cells total P-gp is about 10-fold greater than efflux active P-gp, whereas in MDCKII-hMDR1-NKI cells these values are within twofold. We further visualized the microvilli in MDCKII-hMDR1-NKI and Caco-2 cells using three-dimensional structured illumination super-resolution microscopy and found that the microvilli in Caco-2 cells are taller and more densely packed than those in MDCK-hMDR1-NKI cells. We hypothesized over 10 years ago that only P-gp at the tips of the microvilli contribute significantly to efflux activity, whereas the remaining P-gp are involved in a futile cycle of efflux of amphipathic drugs from the microvillus membrane, followed by their reabsorption into the same or nearby microvillous membranes. The difference between the levels of total and efflux active P-gp in Caco-2 cells can be explained by the more densely packed microvilli in Caco-2 cells, which would lead to a substantial fraction of P-gp not contributing to final release of drug into the apical chamber. Our results suggest that the effect of microvilli morphology differences between in vitro and in vivo systems must be considered when scaling transporter activity for efflux transporters of amphipathic compounds, for example, P-gp.

Like in most Crustacea, the cuticle of terrestrial isopods is hardened by a calcareous mineral phase. This rigid cuticle is frequently shed during a process called moulting. To reduce calcium loss, Porcellio scaber eats the shed cuticle, the exuviae, and absorb the calcium from it through large tubular diverticula of the intestine, called the mid gut glands or hepatopancreas. After moulting the absorbed calcium should be transported immediately into the hemolymph from which it is used to rapidly mineralize the new cuticle. This suggests that the hepatopancreas epithelium transports calcium from the lumen to the hemolymph. We used TEM, energy-filtered TEM and electron-probe X-ray microanalysis to analyse the distribution of elevated calcium within the hepatopancreas cells of P. scaber. We used animals in the postmoult stage that have eaten their exuviae and, as a control, those that have not ingested the exuviae. To minimize calcium loss within the samples, we used high pressure frozen and freeze substituted samples and propane-1-3-diol as floatation medium for thin-sectioning. The results reveal intracellular dense deposits containing calcium, phosphorus and oxygen at the apical microvillus membrane, within the cytoplasm, attached to vesicles and to the basolateral membrane, as well as extracellular between cells and the basal lamina. Control animals were devoid of these deposits. The results indicate that calcium from the exuviae is absorbed and transported across the epithelium into the hemolymph. We propose that during transport, intracellular calcium is bound to phosphate avoiding toxic effects of high concentrations of ionized calcium.

Insulin resistance is present in the majority of patients with non-insulin-dependent diabetes mellitus (NIDDM) and obesity. In this study, we aimed to investigate the key genes and potential molecular mechanism in insulin resistance. Expression profiles of the genes were extracted from the Gene Expression Omnibus (GEO) database. Pathway and Gene Ontology (GO) enrichment analyses were conducted at Enrichr. The protein&ndash, protein interaction (PPI) network was settled and analyzed using the Search Tool for the Retrieval of Interacting Genes (STRING) database constructed by Cytoscape software. Modules were extracted and identified by the PEWCC1 plugin. The microRNAs (miRNAs) and transcription factors (TFs) which control the expression of differentially expressed genes (DEGs) were analyzed using the NetworkAnalyst algorithm. A database (GSE73108) was downloaded from the GEO databases. Our results identified 873 DEGs (435 up-regulated and 438 down-regulated) genetically associated with insulin resistance. The pathways which were enriched were pathways in complement and coagulation cascades and complement activation for up-regulated DEGs, while biosynthesis of amino acids and the Notch signaling pathway were among the down-regulated DEGs. Showing GO enrichment were cardiac muscle cell&ndash, cardiac muscle cell adhesion and microvillus membrane for up-regulated DEGs and negative regulation of osteoblast differentiation and dendrites for down-regulated DEGs. Subsequently, myosin VB (MYO5B), discs, large homolog 2(DLG2), axin 2 (AXIN2), protein tyrosine kinase 7 (PTK7), Notch homolog 1 (NOTCH1), androgen receptor (AR), cyclin D1 (CCND1) and Rho family GTPase 3 (RND3) were diagnosed as the top hub genes in the up- and down-regulated PPI network and modules. In addition, GATA binding protein 6 (GATA6) , ectonucleotide pyrophosphatase/phosphodiesterase 5 (ENPP5), cyclin D1 (CCND1) and tubulin, beta 2A (TUBB2A) were diagnosed as the top hub genes in the up- and down-regulated target gene&ndash, miRNA network, while tubulin, beta 2A (TUBB2A), olfactomedin-like 1 (OLFML1), prostate adrogen-regulated mucin-like protein 1 (PARM1) and aldehyde dehydrogenase 4 family, member A1 (ALDH4A1)were diagnosed as the top hub genes in the up- and down-regulated target gene&ndash, TF network. The current study based on the GEO database provides a novel understanding regarding the mechanism of insulin resistance and may provide novel therapeutic targets.

Organic anion transporters (OATs) and organic anion transporting polypeptides (OATPs) are transport proteins that mediate exchange of metabolites, hormones and waste products. Directional transport by these transporters can occur when exchange is coupled to the gradients of other substrates. This study investigates whether the activity of OATP4A1 and OATP2A1 on the maternal facing microvillus membrane of the placental syncytiotrophoblast is coupled to the glutamate gradient. OAT and OATP transporter proteins were over expressed in Xenopus oocytes to study their transport characteristics. Further transport studies were performed in term human placental villous fragments. Xenopus oocytes expressing OATP4A1 mediated glutamate uptake. No glutamate transport was observed in oocytes expressing OAT1, OAT3, OAT7 or OATP2A1. In oocytes expressing OATP4A1, uptake of estrone sulphate, thyroid hormones T3 and T4 and the bile acid taurocholate stimulated glutamate efflux. In term placental villous fragments addition of estrone sulphate and taurocholate trans-stimulated glutamate efflux. Coupling of OATP4A1 to the glutamate gradient may drive placental uptake of estrone-sulphate and thyroid hormone while also facilitating uptake of potentially harmful bile acids. In contrast, if OATP2A1 is not coupled to a similar gradient, it may function more effectively as an efflux transporter, potentially mediating efflux of prostaglandins to the mother. This study provides further evidence for glutamate as an important counter-ion driving transport into the placenta., Highlights • OATP4A1 and OATP2A1 are present on the maternal facing surface of the placenta. • OATP4A1, but not OATP2A1, activity was coupled to glutamate efflux. • Placental estrone-sulphate and thyroid hormone uptake is coupled to glutamate efflux. • Glutamate acts as a counter-ion for OATP4A1, driving transport into the placenta.

The high demand of the fetus for Docosahexaenoic acid, DHA, is satisfied by a concert of several mechanisms that take place in the placental terminal villi. Scarcity of laboratory data makes the detailed description of these mechanisms elusive. Here, the DHA transport across the placenta is modeled as a boundary value problem that accounts for diffusion, reactions with fatty acids binding proteins, FABPs, and metabolic consumption within the Syncytiotrophoblast, ST. For any given DHA fluxes at the bordering membranes, analytical and numerical solutions yield the DHA concentration profile within the ST. We find that in order to comply with adequate DHA delivery to the fetus and with physiological DHA concentrations in the maternal and fetal circulations, it is essential to have a significant rise of DHA concentration at the microvillus membrane, MVM and a rapid dissociation of the DHA from the FABP. The model establishes the relations between the concentrations of the FABPs in the ST, their equilibrium dissociation constant from the DHA, and the placental DHA metabolic degradation rate, hitherto unknown. We conclude that the bound (to the protein) DHA molecule is degraded at a rate of 0.3–0.45 s−1.

This chapter is devoted to gastric absorption of fatty acids in suckling animals and infants. Conclusive proof was established that medium and short chain fatty acids, particularly caprylic acid in nonruminants, and butyric acid in ruminants, when introduced in the stomach, were disappearing at a relatively high rate. At the relatively low pH of the gastric contents, long chain fatty acids are in the lipophilic undissociated form and, in the absence of bile salts of their critical micellar concentration, they remain solubilized in the oil phase. In the rat, the intestinal microvillus membrane has been reported to contain as much as 38% of total lipids in dry weight, with the following approximate composition: 20% of neutral lipids, 30% of phospholipids, and as much as 50% of glycolipids. The uptake of medium chain fatty acids by everted sacs of rat jejunum exhibits a linear relationship with the bulk phase concentration, thus demonstrating the absence of any saturation kinetics.

Because there is little absorption of orally administered vancomycin hydrochloride (VCM) through the normal intestinal microvillus membrane, the pharmacokinetics of VCM absorbed from the digestive tract are mostly unknown. Here we report a case of severe colitis and renal insufficiency in which the serum concentration of VCM reached the supratherapeutic range after oral administration. A 54-year-old man receiving outpatient chemotherapy for rectal cancer was admitted to our hospital for severe sepsis and acute renal failure. Multimodal therapy including continuous renal replacement therapy (CRRT) and mechanical ventilation was initiated, and oral VCM administration (0.5 g every 6 h) was begun for suspected severe pseudomembranous colitis with large amounts of watery stool. Despite continued CRRT, the serum VCM concentration increased to 30.6 μg/mL after 4 days. Based on pharmacokinetic analysis, the bioavailability of VCM was estimated to be over 54.5%. Colonoscopy showed that the mucosa was severely damaged throughout the large intestine, resulting in considerable exudation of plasma and blood. This case indicates the need for careful and early monitoring during high-dose oral VCM administration to patients with severe mucosal injury and renal insufficiency.

PLAC1 (Placenta-Specific 1) is a recently described, trophoblast-expressed gene essential for normal placental development. The protein localizes to the microvillus membrane surface of the syncytiotrophoblast in direct proximity to the maternal compartment. Although its role has not been defined, increased circulating levels of human PLAC1 mRNA in maternal blood are associated with preeclampsia. Furthermore, PLAC1-null mice exhibit decreased viability in the peripartum period suggesting a role in pregnancy maintenance late in gestation. We examined PLAC1 gene expression in the human placenta during normal pregnancy and pregnancies associated with maternal diabetes and preeclampsia using quantitative, real time PCR (q-RT-PCR). Although there was no apparent difference in PLAC1 gene expression among human pregnancies complicated by diabetes or preeclampsia, an unexpected effect of labor was noted at term. PLAC1 expression in placentae delivered vaginally following induced or spontaneous labor was significantly reduced compared to placentae not exposed to labor making it one of only a few placental genes influenced by labor. The significance of this finding is unknown. Viewed in the context of its importance in placental development, however, these findings are consistent with a role for PLAC1 in the maintenance of the maternal-fetal interface.

Editorial The gastrointestinal tract is the largest exposed surface among mammalian macroorganisms. It is constantly exposed to substantial challenges and exerts a potpourri of diverse physiological functions. Among the major intestinal functions is the digestion of nutrients and subsequent uptake and metabolism of their respective building blocks, control of absorption and secretion of water, salts and nutrients, interaction of the mucosal surface with micro-organisms and cross-talk with the submucosa, and communication at the basolateral surface with cells of innate and adaptive immune system. These functions are tightly linked to a large network of protein and lipid components that exert their function at different stages of intestinal development and differentiation of intestinal cells. Obviously, genetic alterations linking these vital networks elicit malfunctions that are associated with severe disease phenotypes. On September 4 and 5, 2016, a workshop on the molecular biology of the gut was held during the Congress of the German Society for Pediatric and Adolescent Medicine (DGKJ) in Munich and was generously sponsored by QOL Medical LLC, Vero Beach, FL, USA. This was a multidisciplinary workshop that combined molecular, cellular, immunological, and microbiological aspects of the gastrointestinal tract with clinical medicine and nutritional science. Experts viewed the gut as an organ from different angles and presented timely and comprehensive reviews on the impact of intestinal cellular and protein components and the corresponding basic mechanisms that maintain the functional capacities and the physiological hemostasis of the gut. The digestive function in the intestine, for example, is one of the most important and essential processes that are orchestrated by an organized and regulated network of hydrolases and transporters that are located in the microvillus membrane. Unraveling of the structural-functional relationships of these components as well as their biosynthetic and processing pathways has experienced a huge progress in the last few years, so that their malfunctioning and association with the onset of intestinal disorders and symptoms can be clarified. Particularly, the cell biology and pathobiochemistry of intestinal disaccharidases has occupied a major part of this workshop by virtue of the primordial function of the sucraseisomaltase enzyme complex in carbohydrate and human starch digestion to glucose (α-glucogenesis) [1]. Sucraseisomaltase is the major brush border enzyme with almost 5–7 % protein content of the entire brush border membrane and is the enzyme that is responsible for the digestion of almost all disaccharides that are transported to the intestinal lumen [2]. The expression of this protein during cellular differentiation as well as primary and secondary deficiencies has been among the topics discussed in this workshop [3]. Particularly, recent progress in identification of a large number of mutations associated with primary genetically determined sucraseisomaltase deficiency (GSID or CSID) as well as various inheritance forms of this disorder has strongly suggested that this disorder is more common than initially thought. The implications of studying GSID is an important step towards developing therapies and envision approaches to botanical and technological alterations in food that will benefit all populations. Interestingly, the pathogenesis and the basic mechanisms that account for irritable bowel syndrome (IBS), one of the most common intestinal diseases, are until present obscure [4, 5]. Current concepts assume that aberrant or reduced functional capacities of intestinal disaccharidases, such as sucrase-isomaltase, are likely to be linked to the symptoms of IBS. Not only is aberrant function of intestinal disaccharidases associated with chronic diarrhea, but also fructose malabsorption and insufficiencies in glucose transporters or NHE3 [6]. The workshop has also discussed the more global defects at the cellular levels such as in celiac disease, microvillus inclusion disease, or cystic fibrosis which also elicit chronic diarrhea [7–9]. Altogether, these disorders lead to substantial alterations of the intestinal microbiota and may alter and disturb the finely * Correspondence: hassan.naim@tiho-hannover.de Department of Physiological Chemistry, University of Veterinary Medicine Hannover, Bunteweg17, D-30559 Hannover, Germany Full list of author information is available at the end of the article Molecular and Cellular Pediatrics

We have worked out a simplification of the procedure described by Schmitz et al. (Biochim. Biophys. Acta (1973) 323, 98--112) for the preparation of brush border membranes from small intestine. The procedure ultimately adopted is simple, rapid, does not necessarily require scraping and can be started from fresh or frozen material. It can be scaled up easily, allowing a quick production of large amounts of brush border membrane vesicles. These vesicles prove to be excellently suited for transport studies, as suggested by our measurements of D-glucose transport. Using these vesicles, the mode of choline transport across the brush border membrane was also investigated. Choline transport was found to occur by a saturable component with a Km of 83 +/- 4 micrometer (at 20 degrees C) and by a non-saturable component. It is independent of the presence of Na+ and appears to be non-electrogenic.

Enterotoxins of Staphylococcus aureus are among the most common causes of food poisoning. Acting as superantigens they intoxicate the organism by causing a massive uncontrolled T cell activation that ultimately may lead to toxic shock and death. In contrast to our detailed knowledge regarding their interaction with the immune system, little is known about how they penetrate the epithelial barrier to gain access to their targets. We therefore studied the uptake of two staphylococcal enterotoxins (SEs), SEA and SEB, using organ cultured porcine jejunal explants as model system. Attachment of both toxins to the villus surface was scarce and patchy compared with that of cholera toxin B (CTB). SEA and SEB also bound to microvillus membrane vesicles in vitro, but less efficiently than CTB, and the binding was sensitive to treatment with endoglycoceramidase II, indicating that a glycolipid, possibly digalactosylceramide, acts as cell surface receptor at the brush border. Both SEs partitioned poorly with detergent resistant membranes (DRMs) of the microvillus, suggesting a weak association with lipid raft microdomains. Where attachment occurred, cellular uptake of SEA and SEB was also observed. In enterocytes, constitutive apical endocytosis normally proceeds only to subapical early endosomes present in the actomyosin-rich "terminal web" region. But, like CTB, both SEA and SEB penetrated deep into the cytoplasm. In conclusion, the data show that after binding to the enterocyte brush border SEA and SEB perturb the apical membrane trafficking, enabling them to engage in transcytosis to reach their target cells in the subepithelial lamina propria.

The present study describes the in vivo ameliorating effect of Lactobacillus casei supplementation in renourished Giardia intestinalis infected BALB/c mice. It was observed that daily administration of probiotic 7 days prior to Giardia-infection to renourished mice, efficiently reduced the excretion of Giardia cysts and trophozoite counts, along with significant increased fecal lactobacilli counts compared with Giardia-infected mice. It was also observed that oral feeding of probiotic to renourished-Giardia-infected mice abrogated all the anthropometric and biochemical anomalies. Histologically, morphological and cellular alteration of microvillus membrane integrity revealed that probiotic administration further ameliorated the mucosal damage in renourished-probiotic-Giardia-infected mice compared to severe microvillus atrophy, oedematous, vacuolated epithelial cells and ileitis in renourished-Giardia and Giardia-infected mice. Thus, it is suggested that probiotic used as the functional food helps in restoration of anthropometric, biochemical alterations and atrophied gut by enhancing the goblet cells and reducing the giardiasis.

The study describes the in vivo activity of Lactobacillus casei in malnourished Giardia lamblia -infected BALB/c mice. By experimentation, it was found that daily administration of the probiotic 7 days before inoculation with Giardia trophozoites in malnourished mice efficiently reduced both the severity and duration of giardiasis. More specifically, excretion of Giardia cysts and trophozoites counts were reduced, while faecal lactobacilli counts increased significantly in probiotic-fed malnourished mice, compared with control mice. Interestingly, it was also observed that oral feeding of the probiotic to malnourished mice abrogated all the anthropometric and biochemical anomalies. Histologically, morphological and cellular alteration of microvillus membrane integrity revealed that probiotic administration ameliorated the mucosal damage in malnourished, probiotic-inoculated, Giardia-infected mice compared with the severe microvillus atrophy, œdematous and vacuolated epithelial cells, and ileitis in malnourished Giardia-infected mice. The results clearly show the antigiardial effect of the probiotic in vivo by modulating the gut cells to inhibit the colonization and multiplication of Giardia trophozoites, thus reducing the severity and duration of murine giardiasis.

Intestinal lipid transport is a highly evolved and astonishingly efficient process whose genetic basis and metabolic regulation continues to provide surprises. The essence of the process (Figure 1) involves uptake of micellarized dietary long chain fatty acids (FA) and monoglyceride across the microvillus membrane, transport through apical cytoplasmic domains, and enzymatic reassembly into complex lipid droplets within membrane profiles of the endoplasmic reticulum (ER) 1 . These lipid droplets then fuse with a requisite structural protein, apolipoprotein B48 (apoB48), in a process involving physical interaction of the nascent apoB48 protein at its amino terminus with a resident ER chaperone, microsomal triglyceride transfer protein (Mttp), thereby initiating formation of a primordial lipoprotein particle 1 . Further addition of neutral lipid within the ER allows progressive enlargement of the lipoprotein particle, which is surrounded by a single molecule of apoB48 1 . The maturing lipoprotein particles (prechylomicrons) undergo vectorial vesicular transport through Golgi membranes 2 where they undergo terminal modifications and are secreted as mature chylomicron (CM) particles into the lacteal for transport into the mesenteric lymph. Among the continuing mysteries is the nature and identity of the microvillus membrane FA carrier(s) and the role of paracrine and endocrine influences on the dynamics of intestinal lipoprotein assembly and secretion.

The luminal surface of enterocytes is covered with glycocalyx which is rich in glycoproteins. Ethanol ingestion is shown to induce morphological and biochemical changes in the intestine. In this study, the effect of ethanol ingestion on membrane glycoproteins has been investigated. Chemical analysis of microvillus membranes revealed an increase in hexose and sialic acid contents, but a reduction in fucose levels in ethanol-fed rats compared with controls. The observed changes were apparent in animals fed with ethanol for 35-56 days compared with controls. Lectin-binding assay indicated an increase in Wheat germ agglutinin (affinity for GlcNAc/sialic acid) and a decrease in Aleuria aurantia (affinity for alpha-L: -fucose) reactivity of brush borders in ethanol-fed animals for 4-8 weeks. Western blot analysis using biotin-labeled Wheat germ agglutinin revealed increased binding to proteins of M(r) 66-205 kDa in ethanol-fed rats compared with controls. The binding of Aleuria aurantia to membrane proteins of M(r) 97-185 kDa was reduced in ethanol-fed animals. These findings suggest that long-term ethanol feeding modulates the sialylation and fucosylation processes of microvillus membrane proteins in rat intestine. This could affect the intestinal digestive and absorptive functions in chronic alcoholism.

We previously demonstrated that the number and height of oocyte microvilli were reduced in baboon fetuses deprived of estrogen in utero and restored to normal in animals supplemented with estradiol. Phosphorylated ezrin and Na+/H+ exchange regulatory factor 1 (NHERF, now termed SLC9A3R1) link f-actin bundles to the membrane, whereas alpha-actinin cross-links f-actin to form microvilli. Therefore, we determined whether these proteins were expressed in oocytes of the fetal baboon ovary and whether expression and/or localization were altered between mid and late gestation in association with an increase in estrogen and in late gestation in animals in which estrogen was suppressed (>95%) or restored by treatment with an aromatase inhibitor with or without estradiol. Expression of alpha-actinin was low at mid gestation, increased on the surface of oocytes of primordial follicles in late gestation, and was negligible in the ovaries of estrogen-suppressed fetuses and normal in animals treated with estro...

Cryptosporidium parvum mainly invades the intestinal epithelium and causes watery diarrhea in humans and calves. However, the invasion process has not yet been clarified. In the present study, the invasion process of C. parvum in severe combined immunodeficiency (SCID) mice was examined. Infected mice were necropsied; the ilea were double-fixed routinely and observed by scanning and transmission electron microscopy. In addition, the microvillus membrane was observed by ruthenium red staining. Scanning electron micrographs showed elongation of the microvilli at the periphery of the parasite. The microvilli were shown to be along the surface of the parasite in higher magnification. Transmission electron microscopy confirmed that the invading parasites were located among microvilli. Parasites existed in the parasitophorous vacuole formed by the microvillus membrane. The parasite pellicle attached to the host cell membrane at the bottom of the parasite, and then the pellicle and host cell membrane became unclear. Subsequently, the pellicle became complicated and formed a feeder organelle. In addition, invasion of the parasite was not observed in either a microvillus or the cytoplasm of the host cell. Therefore, C. parvum invades among microvilli, is covered with membranes derived from numerous microvilli, and develops within the host cell.

We examined the presence of Zn-transporters (ZnT1, ZnT3, ZnT4, and ZnT6) proteins and zinc ions in rat choroid epithelium with immunohistochemistry and zinc selenide autometallography (ZnSe(AMG)). The four ZnT proteins were all expressed in the choroid epithelial cells. ZnT3 immunostaining was found in vesicle membranes in the apical part of the cells, associated to the microvillus membrane. Correspondingly, the ZnSe(AMG) technique revealed zinc ions in small vesicles, in microvilli, and multivesicular bodies in the epithelial cells. Traceable zinc ions were also found in lysosome-like organelles of fenestrated endothelial cells, but here no corresponding ZnT3 immunostaining was seen. The observations suggests that the choroid plexus is instrumental to regulation of the level of zinc ions in the cerebrospinal fluid.

This review focuses on the regulation of the glycoprotein glycosylation process in small intestine and colon during postnatal development. Glycoproteins play a prominent part in intestine as mucins secreted by the goblet cells and as molecules of biological interest largely present in the microvillus membrane of the enterocytes (digestive enzymes, transporters). The age-related changes in the intestinal glycosylation control the quality of glycan chains of glycoproteins. Postnatal maturation is observed at all stages of the glycoprotein glycosylation. But it is essentially characterised in the external glycosylation by a shift from sialylation to fucosylation depending on the transcriptional regulation of the corresponding glycosyltransferases, but also on coordinate changes in the activities of glycosyltransferases and of their regulatory proteins, in nucleotide-sugar bioavailability and in product degradation by oxidases. Many factors have been evoked to trigger these changes, among which are hormonal (glucocorticoids, insulin) and dietary factors. Changes in the structure of the glycoprotein glycans might be important for the transport, the barrier function, the implantation of the immune defences and of the microflora and even probably for the biological activity of some digestive enzymes.

Intestinal cholesterol absorption is a major determinant of plasma low density lipoprotein-cholesterol (LDL33333391) concentrations. Ezetimibe (SCH 58235) and its analogs SCH 48461 and SCH 58053 are novel potent inhibitors of cholesterol absorption whose mechanism of action is unknown. These studies investigated the effect of SCH 58053 on cholesterol metabolism in female 129/Sv mice. In mice fed a low cholesterol rodent diet containing SCH 58053, cholesterol absorption was reduced by 46% and fecal neutral sterol excretion was increased 67%, but biliary lipid composition and bile acid synthesis, pool size, and pool composition were unchanged. When the dietary cholesterol content was increased either 10- or 50-fold, those animals given SCH 58053 manifested lower hepatic and biliary cholesterol concentrations than did their untreated controls. Cholesterol feeding increased the relative mRNA level for adenosine triphosphate-binding cassette transporter A1 (ABCA1), ABC transporter G5 (ABCG5), and ABC transporter G8 (ABCG8) in the jejunum, and of ABCG5 and ABCG8 in the liver, but the magnitude of this increase was generally less if the mice were given SCH 58053.We conclude that the inhibition of cholesterol absorption effected by this new class of agents is not mediated via changes in either the size or composition of the intestinal bile acid pool, or the level of mRNA expression of proteins that facilitate cholesterol efflux from the enterocyte, but rather may involve disruption of the uptake of luminal sterol across the microvillus membrane.

Escherichia coli strain RDEC-1 causes a diarrheagenic infection in rabbits with AF/R1 fimbriae, which have been identified as an important colonization factor in RDEC-1 adherence leading to disease. The AF/R1-mediated RDEC-1 adherence model has been used as a model systems for E. coli diarrheal diseases. In this study, RDEC-1 adhered specifically to small intestinal brush borders, with both sialic acid and β-galactosyl residues apparently involved. The AF/R1-mediated adherence activity of [ 14 C]-labeled RDEC-1 was analyzed quantitatively by using 24-well plates coated with purified brush borders and purified microvilli. Two microvillus membrane proteins (130 and 140 kDa) were individually isolated, and chicken antibody raised to each protein inhibited bacterial adherence. These same two proteins, previously shown to be recognized by AF/R1, were individually digested with trypsin, and the amino acid sequences of peptides were determined by reversed-phase capillary liquid chromatography-mass spectrometry tandem mass spectrometry (LC-MS). This LC-MS analysis indicated that these proteins are subunits of the rabbit sucrase-isomaltase protein (SI) complex. Guinea pig serum raised to purified rabbit SI complex inhibited bacterial adherence to microvilli. Additionally, as determined by high-performance thin-layer chromatography and autoradiography, RDEC-1 adhered selectively, via AF/R1 fimbriae, to a glycolipid tentatively identified as galactosylceramide (Galβ1-1Cer) in the lipid extract of rabbit small intestinal brush borders. RDEC-1 adherence to Galβ1-1Cer was partially inhibited in the presence of galactose. These combined results indicate that the endogenous receptor molecule for AF/R1 fimbriae of RDEC-1 is each individual component of the SI complex, although binding to glycolipid may be responsible for an additional adherence mechanism.

BACKGROUND—The mechanism of iron absorption by the intestine and its transfer to the main iron storage site, the liver, is poorly understood. Recently an iron carrier was cloned and named DMT1 (divalent metal transporter 1). AIMS—To determine the level of DMT1 gene expression and protein distribution in duodenum and liver. METHODS—A DMT1 cRNA and antibody were produced and used in in situ hybridisation and immunohistochemistry, respectively, in rats in which the iron stores were altered by feeding diets with normal, low, and high iron content. RESULTS—Duodenal DMT1 mRNA was low in crypts and increased at the crypt-villus junction in iron deficient and control rats; it fell in the iron loaded state. Staining for DMT1 protein was not detected in crypts. In villus enterocytes, protein staining was localised to the microvillus membrane in iron deficiency, in the cytoplasm and to a lesser extent in the membrane in controls, and entirely in the cytoplasm of iron loaded animals. Liver DMT1 mRNA was distributed evenly across hepatocytes. DMT1 protein staining was observed on hepatocyte plasma membranes, with highest values in the iron loaded state, lower values in control animals, and none after iron depletion. CONCLUSIONS—Results are consistent with a role for DMT1 in the transmembrane transport of non-transferrin bound iron from the intestinal lumen and from the portal blood. Keywords: DMT1; iron transporters; iron absorption; duodenum; liver

Objective: Altered transport functions in the placenta might contribute to adverse outcome of pregnancies in women with diabetes. Therefore we studied placental glucose transport in this pregnancy complication. Study Design: Syncytiotrophoblast microvillous membrane vesicles and basal membrane vesicles were isolated from women with uneventful pregnancies (control subjects, n=21) and from women with pregnancies complicated by insulin-dependent diabetes mellitus, White class D (n = 7). Glucose uptake and GLUT 1 (glucose transporter 1) expression were studied by means of radiolabeled tracers and Western blot, respectively. Results: In the group with insulin-dependent diabetes mellitus, values for hemoglobin A 1c were moderately elevated in the first trimester (6.61 ± 0.35) but not later in pregnancy and 4 of the 7 neonates were large for gestational age. In the basal membrane vesicles, insulin-dependent diabetes mellitus was associated with a 40% increase in GLUT 1 expression and a 59% higher mediated uptake of d -glucose. No alterations could be demonstrated in microvillus membrane vesicles. Conclusion: Placental glucose transport capacity appears to be increased in insulin-dependent diabetes mellitus. These alterations might explain the occurrence of macrosomia despite well-controlled diabetes. (Am J Obstet Gynecol 1999;180:163-8.)

The mechanisms for regulating the intracellular pH (pHi) level during oligopeptide absorption were investigated in the enterocytes from guinea-pig ileum by identifying the acid-base transporters responsible for extruding H+ that enters the cell through the H+-oligopeptide cotransporter. The pHi level was measured by microfluorometry in an isolated villus tip loaded with the pH-sensitive fluoroprobe 2′7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). The oligopeptide-induced increment in the short-circuit current (Isc) was determined in a mucosal sheet in Ussing chambers. A CO2/HCO3−-buffered solution was used. The superfusion of glycylglycine (Gly-Gly, l0 mM) caused a decrease in pHi level, which returned to the basal level after removing Gly-Gly. This pHi recovery was strongly dependent on extracellular Na+. Amiloride partially inhibited the pHi recovery rate with an IC50 value of 41 μM, the maximum inhibition being approximately 70%. In the presence of amiloride at its maximum concentration (0.3 mM), the addition of 0.6 mM DIDS caused a further decrease, but did not abolish the pHi recovery rate. In the absence of CO2 and HCO3−, the pHi recovery was almost completely abolished by 0.3 mM amiloride. The intracellular H+ accumulation induced by 0.3 mM amiloride or by 0.6 mM DIDS, as estimated from the pHi decrease and buffer capacity, was significantly greater during Gly-Gly superfusion than under resting conditions. The increase in Isc induced by luminal glycylproline was attenuated by either removing serosal Na+ or by adding 0.5 mM amiloride or 0.6 mM DIDS to the serosal side. We conclude that both Na+-dependent, amiloride-sensitive acid extrusion, probably by the Na+-H+ exchanger, and Na+- and HCO3−-dependent, DIDS-sensitive acid extrusion, possibly by the Na+-HCO3− cotransporter, are involved in extruding H+ that enters cells by the H+-oligopeptide cotransport. It is proposed that these acid extrusion (or base loading) mechanisms are present in the basolateral membrane and are important for maintaining oligopeptide absorption, as well as the acid extrusion mechanism in the apical membrane. The intracellular pH (pHi) level is thought to play an important role in a variety of cellular processes, including cellular metabolism, protein synthesis, contractility, ion channel conductivity, and cell cycle control (Madshus, 1988). Therefore, pHi needs to be regulated in a narrow range to maintain cellular functions. In the enterocytes of the small intestine, the uptake of such nutrients as oligopeptides (Thwaites et al. 1993a; Fei et al. 1994), amino acids (Kanai & Hediger, 1992; Thwaites et al. 1995), fatty acids (Harig et al. 1991) and carboxylic acids (Tamai et al. 1995) are mediated by cotransport with H+ or by exchange with OH− or HCO3− across the microvillus membrane. Thus, in the enterocytes, the acid-base regulatory mechanisms are important not only for stabilizing the pHi level, but also for supporting the intestinal absorption of those nutrients. Several relevant membrane acid-base transporter molecules, including the Na+-H+ exchanger (Tse et al. 1991; Bookstein et al. 1994; Yun et al. 1995; Hoogerwerf et al. 1996; Wakabayashi et al. 1997), the Na+-HCO3− cotransporter (Osypiw et al. 1994; Peral et al. 1995; Bernardo et al. 1996; MacLeod et al. 1996), and the Cl−-HCO3− exchanger (Sundaram et al. 1991; Chow et al. 1992) have been demonstrated or suggested to be involved in the enterocytes. However, the role of most of these acid-base transporters in pHi regulation during intestinal absorption of nutrients has yet to be elucidated. In respect of oligopeptide transport in the intestine, it is generally assumed that the sequential action of Na+,K+-ATPase in the basolateral membrane and of the Na+-H+ exchanger in the apical membrane creates an acidic extracellular microclimate, providing an inward electrochemical gradient of H+ which is necessary for H+-coupled oligopeptide absorption (Ganapathy et al. 1994; Adibi, 1997). In support of this, it has been demonstrated in the Caco-2 colonic tumour cell line that the apical membrane Na+-H+ exchanger is mainly responsible for discharging H+ that enters the cells through the H+-oligopeptide cotransporter (Thwaites et al. 1993b, 1994). On the other hand, in native intestinal epithelia, this hypothesis has never been verified by experimental evidence. In addition, it is conceivable that several acid extrusion mechanisms in the basolateral membrane could also be involved in oligopeptide absorption, although this currently remains conjecture. The aim of this study was to identify the acid-base transporters in the ileal enterocytes which are responsible for extruding H+ that enters cells through the H+-coupled oligopeptide transporter. To achieve this, pHi was measured by microfluorometry in a villus isolated from the guinea-pig ileum that had been loaded with pH-sensitive fluorescent dye. The dipeptide-induced short-circuit current (Isc) was also determined in an isolated ileal sheet in Ussing chambers to identify the localization, apical vs. basolateral membrane, of these transporters. Parts of this work have been reported previously (Hayashi & Suzuki, 1996, 1997).

We have previously shown that fetal exposure to ethanol in rats produces both structural and biochemical abnormalities in absorptive enterocytes. Among the indicators of injury are derangements in the expression of lactase-phlorizin hydrolase (LPH), which is an essential enzyme for the assimilation of milk. In an animal model of fetal alcohol syndrome, unsuckled newborn rats prenatally exposed to maternal ethanol revealed a 10- to 15-fold increase in the number of LPH mRNA molecules per absorptive enterocyte, compared with controls (Estrada et al., Alcohol. Clin. Exp. Res. 20:1662-1668, 1996). However, lactase activity per cell was similar in both groups. The aim of this study was to characterize the effect of prenatal exposure to ethanol on the processing of LPH mRNA and protein. RNase protection assays using 3'- and 5'-directed antisense RNA probes revealed that the LPH mRNA from ethanol-exposed pups is full length. However, metabolic labeling, followed by immunoprecipitation using an anti-LPH monoclonal antibody, demonstrated a significant alteration in LPH protein processing. Intestinal explants from 21-day ethanol-exposed fetuses that were chased 30 min after a [35S]methionine pulse showed greater amounts of newly synthesized LPH precursors (205 and 220 kDa) and low molecular weight degradation products than controls. However, despite the increases in LPH precursor, the amount of 130 kDa mature LPH was similar in ethanol-exposed and control explants. These data suggest an increase in intracellular degradation of LPH precursor in rats prenatally exposed to ethanol, which occurs before its insertion into the microvillus membrane. Biosynthesis of LPH appears to be upregulated at the transcriptional level, which overcomes the degradation of LPH precursor during processing.

In ileal absorptive cells, carbachol inhibits NaCl absorption and its component brush border Na+/H+ exchanger, acting via basolateral membrane receptors. This carbachol effect involves (i) activation of brush border phosphatidylinositol 4,5-bisphosphate-specific phospholipase C (PLC) activity and brush border but not basolateral membrane translocation of PLC-gamma1 (Khurana, S., Kreydiyyeh, S., Aronzon, A., Hoogerwerf, W. A., Rhee, S. G., Donowitz, M., and Cohen, M. E. (1996) Biochem. J. 313, 509-518); and (ii) brush border tyrosine kinase(s) because mucosal but not serosal addition of the tyrosine kinase inhibitor genistein prevents the carbachol-induced inhibition of NaCl absorption and brush border Na+/H+ exchange. In the present work we identify a pool of villin (a brush border actin-binding protein) in the microvillus membrane fraction of rabbit ileum; this pool of villin is tyrosine-phosphorylated and associates with brush border membrane PLC-gamma1. Villin is present both in the Triton X-100-soluble and -insoluble fractions of the brush border. The Triton X-100-soluble pool is approximately 4-fold smaller than the brush border pool of villin that is present in the Triton X-100-insoluble fraction. Only the villin present in the Triton X-100-soluble fraction of ileal villus brush border associates with PLC-gamma1 and is tyrosine-phosphorylated. Carbachol increases the tyrosine phosphorylation of villin rapidly (as early as 30 s) and transiently. Carbachol also increases the amount of tyrosine-phosphorylated villin that associates with PLC-gamma1. These studies demonstrate that carbachol effects on NaCl absorption are accompanied by an increase in brush border PLC-gamma1 association with villin and an increase in tyrosine phosphorylation of villin. To study the role of cytoskeletal rearrangement in carbachol-induced inhibition of NaCl absorption, we used the F-actin stabilizing drug jasplakinolide. Jasplakinolide prevents the carbachol inhibition of ileal NaCl absorption. This suggests that F-actin severing is necessary for carbachol to inhibit ileal villus NaCl absorption. Since villin is known to sever actin, these studies suggest a role for villin in the signaling cascade that begins at the basolateral membrane with carbachol binding to its receptor and ends at the apical membrane in inhibition of NaCl absorption.

N-benzoyl-L-tyrosyl-p-aminobenzoic acid hydrolase (PPH, human meprin) is a member of the astacin family of Zn-metalloendopeptidases and is highly expressed in the microvillus membrane of human small intestinal epithelial cells. It is a type I transmembrane protein consisting of differentially processed glycosylated alpha and beta subunits. Biosynthesis experiments using transfected, metabolically labelled simian virus 40 (SV40) transformed african green monkey kidney cells (COS-1) and Madin Darby canine kidney (MDCK) cells, have previously shown that PPH alpha was retained in the endoplasmic reticulum (ER) and that for subsequent secretion removal of the alpha-tail was necessary [Grünberg, J., Dumermuth, E., Eldering, J. A.Sterchi, E. E. (1993) FEBS Lett. 335, 376-379]. We proposed an involvement of the alpha-tail in ER retention. To investigate the possible role of the transmembrane and/or the C-terminal domain of the alpha-subunit, tailswitch mutants were constructed in which these domains were exchanged between the alpha and beta subunits. Biosynthesis and post-translational processing of these mutants were investigated in transiently transfected COS-1 cells. The beta/alpha tailswitch mutant, in which the transmembrane and C-cytosolic parts of PPH beta were substituted by the corresponding parts of the PPH alpha subunit, was transported much slower compared with the wild-type PPH beta subunit. In addition, fusion of the alpha-tail to a C-terminally truncated secretory form of PPH alpha leads to its retention in the ER. This mutant, but not the secretory form, coimmunoprecipitated with calnexin, indicating an involvement of this molecular chaperone in retaining PPH alpha in the ER. The alpha/beta tailswitch mutant, in which the transmembrane domain and the C-cytosolic part of PPH alpha were substituted by the corresponding parts of PPH beta, was processed less efficiently in comparison with PPH alpha, resulting in a lower secretion rate. Taken together these data suggest a role of the alpha-tail in mediating association with ER-resident machinery, facilitating C-terminal processing.

Lactulose is one of the most frequently utilised agents in the treatment of constipation and hepatic encephalopathy because of its efficacy and good safety profile. The key to understanding the possible modes of action by which lactulose achieves its therapeutic effects in these disorders lies in certain pharmacological phenomena: (a) lactulose is a synthetic disaccharide that does not occur naturally; (b) there is no disaccharidase on the microvillus membrane of enterocytes in the human small intestine that hydrolyses lactulose; and (c) lactulose is not absorbed from the small intestine. Thus, the primary site of action is the colon in which lactulose is readily fermented by the colonic bacterial flora with the production of short-chain fatty acids and various gases. The purpose of this review is to focus on some pertinent basic aspects of the clinical pharmacology of lactulose and to discuss the possible mechanisms by which lactulose benefits patients with constipation and hepatic encephalopathy.

Insecticidal crystal proteins (ICPs) from Bacillus thuringiensis have been used as biopesticides for the last 35 years. B. thuringiensis is a gram-positive bacterium which produces proteinaceous inclusions during sporulation; these inclusions can be distinguished as distinctively shaped crystals by phase-contrast microscopy. The inclusions are composed of proteins known as ICPs, Cry proteins, or d-endotoxins, which are highly toxic to a wide variety of important agricultural and healthrelated insect pests as well as other invertebrates. Due to their high specificity and their safety for the environment, ICPs are a valuable alternative to chemical pesticides for control of insect pests in agriculture and forestry and in the home. It has been proposed that the rational use of B. thuringiensis toxins will provide a variety of alternatives for insect control and for coping with the problem of insect resistance to pesticides. Intensive screening programs have identified strains of B. thuringiensis from soil samples, plant surfaces, dead insects, and stored grains from all over the world. The isolated strains show a wide range of specificity against different insect orders (Lepidoptera, Diptera, Coleoptera, Hymenoptera, Homoptera, Phthiraptera or Mallophaga, and Acari) and other invertebrates (Nemathelminthes, Platyhelminthes, and Sarcomastigorphora) (13). Currently 45 different serotypes have been catalogued, representing a total number of 58 serovars (28). Many of the ICP genes have been cloned, sequenced, and classified as cry and cty genes. The first classification was based on insecticidal activity (23), with the different Cry proteins denoting ICPs toxic to various insect and invertebrate groups as follows: CryI toxic to lepidopterans, CryII toxic to lepidopterans and dipterans, CryIII toxic to coleopterans, CryIV toxic to dipterans, and CryV and CryVI toxic to nematodes (14). Novel cry genes isolated recently have created some problems for this classification scheme, especially genes that were homologous to known genes but displayed different specificities and genes that had dual specificity. Recently, a novel nomenclature has been proposed based exclusively on amino acid identity (7). To date, over 50 cry gene sequences have been determined and classified into 15 families (7). The cry genes code for proteins with a range of molecular masses from 50 to 140 kDa. Upon ingestion by the susceptible target, the protoxins are solubilized and proteolytically processed to release the toxic fragment (23). During proteolytic activation, peptides are removed from both aminoand carboxyl-terminal ends of the protoxin. For the 130to 140-kDa protoxins, the carboxyl-terminal proteolytic activation removes half of the molecule, resulting in an active toxin fragment of 60 to 70 kDa. A generally accepted model for Cry toxin action is that it is a multistage process. First, the activated toxin binds to receptors located on the apical microvillus membrane of epithelial midgut cells (6, 22, 49). After the toxin binds the receptor, it is thought that there is a change in the toxin conformation allowing toxin insertion into the membrane. Oligomerization of the toxin follows, and this oligomer then forms a pore that leads to osmotic cell lysis (26a, 30, 32, 40). Receptor binding is a key factor in specificity. Two different insect proteins have been identified as receptors for Cry toxins, the 120-kDa aminopeptidase N Cry1Ac toxin-binding protein purified from brush border vesicles of Manduca sexta, Heliothis virescens, and Lymantria dispar (20, 25, 41, 48) and the 210-kDa cadherin-like glycoprotein Cry1Ab toxin-binding protein purified from M. sexta membranes (47). Specific binding involves two steps, one that is reversible and one that is irreversible. Recent data suggest that toxicity correlates with irreversible binding (31). Irreversible binding might be related to insertion of the toxin into the membrane but could also reflect a tighter interaction of the toxin with the receptor. The crystal structures of Cry3A (coleopteran-specific) and Cry1Aa (lepidopteran-specific) toxins have been reported (21, 30). The Cry3A protoxin has a molecular mass of 70 kDa and does not contain the large carboxyl-terminal extension contained in the Cry1Aa toxin. The crystal structure of Cry1Aa toxin was determined from the activated toxin fragment. Both toxins share 36% amino acid identity, and the two structures show high overall similarity (21). Both are globular molecules containing three distinct domains connected by single linkers. Domain I extends from residues 33 to 253 in Cry1Aa and from residues 58 to 290 in Cry3A; it is a seven a-helix bundle in which a central helix (helix a-5) is completely surrounded by six outer helices (Fig. 1A). This domain has been implicated in the channel formation in the membrane. The six a-helices are amphipathic and are long enough to span the 30-A-thick hydrophobic region of a membrane bilayer. Point mutations in the region encoding the central a-5 helix of the Cry1Ac toxin (residues 163 to 170) drastically affect toxicity without affecting binding to larval midgut vesicles (52). Residues 265 to 461 in Cry1Aa and 291 to 500 in Cry3A form domain II (Fig. 1B). Domain II consists of three antiparallel b-sheets with similar topologies packed around a hydrophobic core. This domain represents the most divergent part in structure between the two toxin molecules (21). This domain has been described as the specificity-determining domain, since reciprocal hybrid genes between closely related toxins * Mailing address: Department of Microbiology, Instituto de Biotecnologia, Universidad Nacional Autonoma de Mexico, Apdo. Postal 510-3, Cuernavaca, Morelos, Mexico. Phone: (52) (5) 622-7635. Fax: (52) (73) 17-2388. E-mail: bravo@ibt.unam.mx.

N-Benzoyl-L-tyrosyl-p-aminobenzoic acid hydrolase (PPH, human meprin), is a peptidase found in the microvillus membrane of human small intestinal epithelial cells. PPH belongs to the astacin family of zinc-metalloendopeptidases and is a protein complex composed of two glycosylated subunits, alpha and beta. The present report describes the cloning of the complete beta subunit and the remaining N2-terminal end of the alpha subunit for analysis of their primary structures in addition to the examination of their biogenesis in transfected cell cultures. The complete open reading frame of the PPH beta cDNA translates into 700 amino acid residues compared with 746 residues for the PPH alpha cDNA. The primary structure of beta and alpha subunits are 44% identical and 61% similar. As predicted from their primary structure, the two subunits of PPH have identical modular structures; starting at the N2-terminus both contain a signal peptide, a propeptide, a protease domain containing the astacin signature, a meprin A5 protein tyrosine phospatase mu (MAM) and a meprin and TRAF homology domain (MATH) domain, an epidermal growth factor(EGF)-like domain, a putative transmembrane anchor domain and a short cytosolic tail. Pulse/chase labelling and immuno-Gold electronmicroscopy of recombinant PPH beta and alpha subunits expressed in transfected Madin-Darby canine kidney (MDCK) cells show that post-translational processing and transport of the two subunits are very different. When expressed alone, the beta subunit acquired complex glycan residues, readily formed homodimers and was transported to the plasma membrane. Small amounts of PPH beta were found in the culture medium. In contrast, the cell-bound alpha subunit, when expressed alone, remained primarily in the high-mannose form, was aggregated and not expressed at the cell surface. However, the bulk of mostly endo-beta-N-acetylglucosaminidase H-resistant alpha subunit was found in the filtered culture medium. The proteolytic event that leads to the formation of this soluble transport-competent form occurs in the endoplasmic reticulum (ER). Coexpression of the alpha subunit with the beta subunit allowed the localisation of the alpha subunit to the plasma membrane. These studies indicate that assembly of the two subunits of PPH is required for the localisation of the alpha subunit to the plasma membrane. In contrast to rodent meprin, both PPH subunits are apically secreted from MDCK cells.

The effect of dietary fats on the chemical composition and enzyme activities has been studied in intestinal brush border membranes (BBM) or rats. Animals were given commercial rat pellet diet (RP) or semisynthetic diet rich in either saturated [coconut oil (CCO))] or polyunsaturated [n-6, corn oil (CO) or n-3, fish oil (FO)] fat at the 10% level for 5 weeks. The membrane cholesterol/phospholipid ratio was augmented in CO- or RP-fed rats. There was an increase in level of saturated fatty acids in BBM from CCO- or FO-fed animals. n-3 polyunsaturated fatty acid content was raised in FO-fed rats, while the proportion of linoleic acid and arachidonic acid was enhanced in animals given a CO diet. Membrane fluidity was in the order of CCO < RP = CO < FO. The membrane hexose content was high (p < 0.05) in the CCO group. Hexosamines were elevated (p < 0.05) in CCO- or FO-fed rat brush borders. Membrane fucose was unaltered, while sialic acid content was elevated in CO- (p < 0.05) and FO- (p < 0.01) fed vs. CCO-fed rats. Lectin binding to brush borders corroborated these findings. The activities of alkaline phosphatase, sucrase and lactase were augmented (p < 0.001) in CCO-fed animals. Leucine-aminopeptidase and sucrase activities were depressed by FO feeding. The activities of PNP-beta-glycosidases were the highest in FO-fed rats. These results indicate that dietary fat quality markedly affects microvillus membrane lipid composition, glycosylation and enzyme functions in rat intestine.

Background & Aims: Lactase-phlorizin hydrolase (LPH) is an intestinal microvillus membrane glycoprotein that hydrolyzes lactose and phlorizin. These enzymatic activities have been assigned to glutamic acid (E) residues 1271 and 1747 in rabbit LPH. The aim of this study was to determine directly if this assignment was correct and if these two amino acids are the only nucleophiles required for LPH enzyme activity. Methods: Site-directed mutagenesis of a full-length rat LPH complementary DNA was used to convert the rat homologues E1274 and E1750 to aspartic acid or glycine. Mutants were analyzed by enzyme activity assays. Results: All tested activities of E1274D and E1274G were virtually unaffected. In contrast, mutations E1750D and E1750G resulted in total loss of lactase and cellobiose activities, leaving only low ONP-glc and ONP-gal hydrolase activities detectable. A double mutant containing both E1274G and E1750G had no activity. Conclusions : These studies directly confirm that the two previously identified glutamic acids are essential to the enzymatic activity of rat LPH. Rat lactase activity is not associated with the E1274 site. This study provides the first evidence that rat LPH has its major catalytic site at E1750, representing all of the lactase and the majority of the phlorizin hydrolase activity.

We report the influence of media lipids, growth in lipid-poor medium, and cell differentiation on delta 9, delta 6, and delta 5 desaturase activity in the human CaCo-2 enterocyte cell line. We also describe the level of incorporation of palmitic (16:0), linoleic (18:2n-6), and eicosapentaenoic (EPA) acids (20:5n-3) and their higher homologues into cytosolic and membrane lipids during long-term (10 days) medium supplementation in fully differentiated 16- to 18-day-old cultures. CaCo-2 monolayers reached confluency by day 6 with subsequent development of microvilli and maximal expression of microvillus membrane sucrose, alkaline phosphatase, and gamma-glutamyltransaminase occurring between days 16 and 23 after plating. There was evidence of the presence and modulation of delta 9, delta 6, and delta 5 desaturase activity (delta 9 > delta 6 > delta 5). delta 6 Desaturase activity decreased approximately 2-fold between days 6 and 24 of culture and when the fetal bovine serum concentration was increased from 0.5% to 25%; in contrast, when cells were starved for 72 h, activity increased 5.4-fold. When the media was supplemented with either linoleic acid and/or EPA, both delta 6 and delta 5 desaturase activities were inhibited, the greatest reduction of delta 5 desaturase activity occurring with EPA. Incorporation of media fatty acids plus their desaturase and elongase products was highly dependent on medium composition with the homologues of delta 9 > delta 6 > delta 5. Supplementation of cellular media with 100 microM EPA for 10 days decreased membrane phosphatidylethanolamine arachidonic acid level from 13.2 to 8.9%. From these results we conclude that enterocyte membrane fatty acid composition and desaturase enzyme activity are regulated by both dietary fat intake and cell maturation. The clinical relevance of these observations on lipid dietary modification for the management of chronic inflammatory bowel disease is still uncertain but these observations suggest that the beneficial effects of EPA supplements on human ulcerative colitis may be due to a reduction in enterocyte arachidonic acid content by down-regulation of delta 6 and delta 5 desaturase activity.

The potency and nature of the inhibitory effect of various cationic drugs on the transport of choline across the placental syncytial microvillus membrane was investigated. Tetraethylammonium, a model substrate for organic cation transport, was a poor inhibitor. Enlarging the degree of alkylation of the quaternary ammonium increased the inhibitory effect, in proportion with increasing lipophilicity. Log concentration vs % control uptake curves showed marked differences in inhibitory potency for the different cationic drugs. Hemicholinium-3 inhibited mediated choline uptake in the micromolar range, whereas atropine and mepiperphenidol were less potent. The H2-receptor antagonists cimetidine, ranitidine, and famotidine inhibited choline uptake in the millimolar ranges. Dixon analysis revealed a competitive nature of inhibition for hemicholinium-3 and atropine (Ki = 40 microM and 1.2 mM, respectively). Cimetidine interacted noncompetitively (Ki = 3.4 mM). Since relatively high concentrations were needed to reach half maximal inhibition, impairment of fetal choline supply due to maternal drug use during pregnancy is not to be expected.

Alterations in the distribution of vasoactive intestinal polypeptide (VIP) in normal and ischemic small intestines of dogs were studied by using conventional transmission electron microscope, and immunohistochemistry for light and electron microscopy. At the light microscopic level, immunoreactivity was evident in the intestinal ganglionic cells of control segments. At the electron microscopic level using a pre-embedding method, the entire cytoplasm of the ganglionic cells in the control segments was filled with VIP immunoreactive products, while the post-embedding experiment showed positive reactions only within the VIP granules and Golgi vesicles. After 30 min of ischemia, immunoreactivity was greatly decreased in the ganglionic cells and a large amount of VIP immunoreactive product appeared in the striated border of epithelial cells and in nerve fibers of the subepithelial layer. These results suggest that intestinal ischemia might lead to the release of VIP, which seems to bind to the microvillus membrane of epithelial cells. The relationship between the changes in VIP distribution and its protecting mechanisms of ischemic damage is discussed.