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We hypothesized that duodenal [HCO.sup.-.sub.3] secretion alkalinizes the microclimate surrounding intestinal alkaline phosphatase (IAP), increasing its activity. We measured AP activity in rat duodenum in situ in frozen sections with the fluorogenic substrate ELF-97 phosphate and measured duodenal [HCO.sup.-.sub.3] secretion with a pH-stat in perfused duodenal loops. We examined the effects of the IAP inhibitors L-cysteine or L-phenylalanine (0.1-10 mM) or the tissue nonspecific AP inhibitor levamisole (0.1-10 mM) on AP activity in vitro and on acid-induced duodenal [HCO.sup.-.sub.3] secretion in vivo. AP activity was the highest in the duodenal brush border, decreasing longitudinally to the large intestine with no activity in stomach. Villous surface AP activity measured in vivo was enhanced by [PGE.sub.2] intravenously and inhibited by luminal L-cysteine. Furthermore, incubation with a pH 2.2 solution reduced AP activity in vivo, whereas pretreatment with the cystic fibrosis transmembrane regulator (CFTR) inhibitor [CFTR.sub.inh]-172 abolished AP activity at pH 2.2. L-Cysteine and L-phenylalanine enhanced acid-augmented duodenal [HCO.sup.-.sub.3] secretion. The nonselective P2 receptor antagonist suramin (1 mM) reduced acid-induced [HCO.sup.-.sub.3] secretion. Moreover, L-cysteine or the competitive AP inhibitor glycerol phosphate (10 mM) increased [HCO.sup.-.sub.3] secretion, inhibited by suramin. In conclusion, enhancement of the duodenal [HCO.sup.-.sub.3] secretory rate increased AP activity, whereas inhibition of AP activity increased the [HCO.sup.-.sub.3] secretory rate. These data support our hypothesis that [HCO.sup.-.sub.3] secretion increases AP activity by increasing local pH at its catalytic site and that AP hydrolyzes endogenous luminal phosphates, presumably ATP, which increases [HCO.sup.-.sub.3] secretion via activation of P2 receptors. duodenum; brush-border membrane; ELF-97 phosphate

The transporter PepT1, apically expressed in intestinal epithelial cells, is responsible for the uptake of di/tripeptides. PepT1 is also expressed in nonpolarized immune cells. Here we investigated the localization of PepT1 in lipid rafts in small intestinal brush border membranes (BBMs) and polarized and nonpolarized cells, as well as functional consequences of the association of PepT1 with lipid rafts. Immunoblot analysis showed the presence of PepT1 in low-density fractions isolated from mouse intestinal BBMs, polarized intestinal Caco2-BBE cells, and nonpolarized Jurkat cells by solubilization in ice-cold 0.5% Triton X-100 and sucrose gradient fractionation. PepT1 colocalized with lipid raft markers GM1 and N-aminopeptidase in intestinal BBMs and Caco2-BBE cell membranes. Disruption of lipid rafts with methyl-[beta]-cyclodextrin (M[beta]CD) shifted PepT1 from low- to high-density fractions. Remarkably, we found that M[beta]CD treatment increased PepT1 transport activity in polarized intestinal epithelia but decreased that in intestinal BBM vesicles and nonpolarized immune cells. Mutational analysis showed that phenylalanine 293, phenylalanine 297, and threonine 281 in transmembrane segment 7 of the human di/tripeptide transporter, hPepT1, are important for the targeting to lipid rafts and transport activity of hPepT1. In conclusion, the association of PepT1 with lipid rafts differently modulates its transport activity in polarized and nonpolarized cells. brush border; Caco2-BBE; mice

In the mammalian small intestine, coupled NaCl absorption occurs via the dual operation of Na/H and Cl/HC[O.sub.3] exchange on the villus cell brush border membrane (BBM). Although constitutive nitric oxide (cNO) has been demonstrated to alter gastrointestinal tract functions, how cNO may specifically alter these two transporters to regulate coupled NaCl absorption is unknown. In villus cells, inhibition of cNO synthase (cNOS) with L-[N.sup.G]-nitroarginine methylester (L-NAME) stimulated Na/H exchange whereas Cl/HC[O.sub.3] exchange was unaffected. In villus cell BBM vesicles (BBMV) prepared from rabbits treated with L-NAME, Na/H exchange was also stimulated. D-NAME, an inactive analog of L-NAME, and [N.sup.6]-(1-imonoethyl)-L-lysine dihydrochloride, a more selective inhibitor of inducible NO synthase, did not affect Na/H exchange. Kinetic studies demonstrated that the mechanism of stimulation is secondary to an increase in the maximal rate of uptake of Na, without an alteration in the affinity of the transporter for Na. Northern blot studies demonstrated an increase in the message for the BBM Na/H exchanger NHE3, and Western blot studies showed that the immunoreactive protein levels of NHE3 was increased when cNOS was inhibited. Thus these results indicate that cNO under nominal physiological states most likely maintains an inhibitory tone on small intestinal coupled NaCl absorption by specifically inhibiting BBM Na/H expression. nitro-L-arginine methyl ester; [N.sup.6]-(1-imonoethyl)-L-lysine dihydrochloride; nitric oxide; NHE3; NH/E exchange

SGLT 1 (SLC5A 1) mediates a part of glucose and galactose reabsorption in the mammalian proximal tubule (PT), but the detailed localization of the transporter along the tubule is still disputable. Here, we used several methods to localize rat SGLT1 (rSGLT1) in the kidneys of intact and variously treated male (M) and female (F) rats. In immunoblots of isolated cortical (C) and outer stripe (OS) brush-border membranes (BBM), a peptide-specific polyclonal antibody for rSGLT1 labeled a sharp inzone-, and gender-dependent ~40-kDa protein and a broad ~75-kDa band that exhibited strong zonal (OS > C) and gender differences (F > M). In tissue cryosections, the antibody strongly stained BBM of the $3 PT segments in the OS and medullary rays (F > M) and smooth muscles of the blood vessels and renal capsule (F ~ M) and weakly stained the apical domain of other PT segments in the C (F ~ M). The phlorizin-sensitive uptake of D-[[sup.3]H]galactose in BBM vesicles, as well as the tissue abundance of rSGLT1-specific mRNA, matched the immunoblotting data related to the 75-kDa protein and the immunostaining in S3, proving zonal and gender differences in the functional transporter. Ovariectomy had no effect, castration upregulated, whereas treatment of castrated rats with testosterone, but not with estradiol or progesterone, downregulated the 75-kDa protein and the immunostaining in S3. We conclude that in the rat kidney, the expression of SGLT1 is represented by a 75-kDa protein localized largely in the PT S3 segments, where it exhibits gender differences (F > M) at both the protein and mRNA levels that are caused by androgen inhibition. brush-border membrane; immunocytochemistry: kidney; sex differences; sodium-glucose cotransporters: steroid hormones

The pig small intestinal brush border is a glycoprotein- and glycolipid-rich membrane that functions as a digestive/absorptive surface for dietary nutrients as well as a permeability barrier for pathogens. The present work was performed to identify carbohydrate-binding (lectinlike) proteins associated with the brush border. Chromatography on lactose-agarose was used to isolate such proteins, and their localization was studied biochemically and by immunofluorescence microscopy and immunogold electron microscopy. IgG and IgM were the two major proteins isolated, indicating that naturally occurring anti-glycosyl antibodies are among the major lectinlike proteins in the gut. IgG and IgM as well as IgA were localized to the enterocyte brush border, and a brief lactose wash partially released all three immunoglobulins from the membrane, indicating that anti-glycosyl antibodies constitute a major part of the immunoglobulins at the lumenal surface of the gut. The antibodies were associated with lipid rafts at the brush border, and they frequently (52%) coclustered with the raft marker galectin 4. A lactose wash increased the susceptibility of the brush border toward lectin peanut agglutin and cholera toxin B, suggesting that anti-glycosyl antibodies compete with other carbohydrate-binding proteins at the lumenal surface of the gut. Thus anti-glycosyl antibodies constitute a major group of proteins associated with the enterocyte brush border membrane. We propose they function by protecting the lipid raft microdomains of the brush border against pathogens. immunoglobulins; enterocyte

Regulation of oligopeptide transporter (Pept-1) in experimental diabetes. Am J Physiol Gastrointest Liver Physiol 283: G133-G138, 2002; 10.1152/ ajpgi.00445.2001.--The knowledge of expression and biology of the intestinal oligopeptide transporter (Pept-1) in a metabolic disorder such as diabetes may have nutritional and pharmacological implications. To study this problem, rats were made diabetic by streptozotocin injection, and Western and Northern blot analyses and nuclear run-on assay were used to determine the protein and gene expressions of Pept-1 and its rate of transcription, respectively. Uncontrolled diabetes for 96 h increased the activity of Pept-1 in the brush-border membrane of intestinal mucosa. Studies of Michaelis-Menten constant, maximal velocity, and protein expression of Pept-1 indicated that an increase in the abundance of this transporter was responsible for the increased activity. Studies of the gene expression showed that uncontrolled diabetes increased the abundance of mRNA encoding Pept-1 without altering its rate of transcription. Lastly, studies of the specificity of the above effect showed that uncontrolled diabetes similarly affected the protein and gene expressions of Pept-1 located in the kidney. In conclusion, the data show that 1) uncontrolled diabetes has a tropic effect on Pept-1 and 2) the effect is systemic, and its molecular mechanism appears to be an increase in the stabilization of mRNA encoding Pept-1. brush-border membrane; gene expression; gene transcription; intestinal transport; renal transport

Diurnal rhythm of [H.sup.+]-peptide cotransporter in rat small intestine. Am J Physiol Gastrointest Liver Physiol 283: G57-G64, 2002; 10.1152/ajpgi.00545.2001.--In mammals, most physiological, biochemical, and behavioral processes show a circadian rhythm. In the present study, we examined the diurnal rhythm of the [H.sup.+]-peptide cotransporter (PEPT1), which transports small peptides and peptide-like drugs in the small intestine and kidney, using rats maintained in a 12-h photo-period with free access to chow. The transport of [[sup.14]C]glycylsarcosine (Gly-Sar), a typical substrate for PEPT1 by in situ intestinal loop and everted intestine, was greater in the dark phase than the light phase. PEPT1 protein and mRNA levels varied significantly, with a maximum at 2000 and minimum at 800. Similar functional and expressional diurnal variations were observed in the intestinal [Na.sup.+]-glucose cotraporter (SGLT1). In contrast, renal PEPT1 and SGLT1 showed little diurnal rhythmicity in protein and mRNA expression. These findings indicate that the intestinal PEPT1 undergoes diurnal regulation in its activity and expression, and this could affect the intestinal absorption of dietary protein. kidney; intestinal absorption; brush-border membranes

Soumounou, Youssouf, Claude Gauthier, and Harriet S. Tenenhouse. Murine and human type I Na-phosphate cotransporter genes: structure and promoter activity. Am J Physiol Renal Physiol 281: F1082-F1091, 2001. First published August 15, 2001; 10.1152/ajprenal.00092.2001.--Na-phosphate ([P.sub.i]) cotransporters in the apical membrane of renal proximal tubular cells play a major role in the maintenance of [P.sub.i] homeostasis. Although two such cotransporters, Npt1 and Npt2, have been identified, little is known about the function and regulation of Npt1. We cloned and characterized the murine (Npt1) and human (NPT1) genes, isolated the 5'-flanking region of Npt1, and analyzed its promoter activity. Npt1 is ~29 kb with 12 exons, whereas NPT1 is ~49 kb with one additional exon. The Npt1 promoter has a TATA-like box but no CAAT box, and the transcription start site was identified by primer extension and 5'-rapid amplification of cDNA ends. Transfection of opossum kidney cells with Npt1 promoter-reporter gene constructs demonstrated significant activity in a 570-bp fragment that was completely inhibited by cotransfection with the transcription factor, hepatocyte nuclear factor (HNF)-3[beta]. Deletion of 200 bp from the 3'-end of the 570-bp fragment abrogated its promoter activity. In addition, promoter activity of a 4.5-kb fragment, but not the 570-bp fragment, was stimulated fourfold by cotransfection with HNF-1[alpha]. Other well-characterized cis-acting elements were identified in the Npt1 promoter. We suggest that Npt1 expression is transcriptionally regulated and provide a basis for the investigation of Npt1 function by targeted mutagenesis. kidney; brush-border membrane; hepatocyte nuclear factor; transcription Received 20 March 2001; accepted in final form 9 August 2001

Research was conducted to examine the toxicity, binding and permeability of four Bacillus thuringiensis Cry1 toxins using brush border membrane vesicles from Spodoptera frugiperda and Spodoptera exigua. Results provide evidence that the toxicities of the four toxins differed significantly. Findings also indicate that there was no relationship between toxicity and dissociation of bound toxins from brush border membrane vesicles.

The effect of purified squalamine, a cationic steroid found in the tissues of the dogfish shark Squalus acanthias, on cloned rabbit Na+/H+ exchanger isoforms NHE1, NHE2 and NHE3 that were transfected in PS120 fibroblasts was investigated. Squalamine was found to decrease the maximal velocity of NHE3 and increase K'[H+]i, but did not affect NHE1 or NHE2 function. Results also showed that the inhibitory effect of squalamine is time dependent, fully reversible and requires the COOH-terminal 76 amino acids of NHE3.

Ileal proglucagon gene expression and postprandial plasma concentrations of proglucagon-derived peptides are reported to change with the type and quantity of dietary fiber ingested by rats. Within the intestine, proglucagon encodes several proglucagon-derived peptides known to modulate intestinal absorption capacity and pancreatic insulin secretion. To determine whether the chronic ingestion of fermentable dietary fiber regulates the expression and synthesis of proglucagon-derived peptides in the distal intestine to modulate glucose homeostasis, the following study was conducted: 16 adult dogs (23 [+ or -] 2 kg) were fed isoenergetic, isonitrogenous diets containing a mixture of high fermentable dietary fibers (HFF) or low fermentable (LFF) wood cellulose for 14 d in a randomized cross-over design. Food was withheld for 16 h before an oral glucose tolerance test was conducted supplying 2 g of glucose/kg body wt, and peripheral blood was collected via a hind-leg catheter at 0, 15, 30, 45, 60, 90 and 120 min for plasma glucose, insulin and glucagon-like peptide-1(7-36)N[H.sub.2] (GLP-1) analyses. Intestinal samples were collected after the second dietary treatment. Ileal proglucagon mRNA, intestinal (GLP-1) concentrations and the integrated area under the curves (AUC) for plasma GLP-1 and insulin were greater and plasma glucose AUC was reduced when dogs were fed the HFF diet compared to the LFF diet (P < 0.05). Intestinal villi heights, brush border and basolateral glucose transporter protein abundance and jejunal transport capacities were significantly greater when dogs were fed the HFF diet than when fed the LFF diet. In conclusion, improvements in glucose homeostasis are observed in healthy dogs when they ingest fermentable fibers. KEY WORDS: * dogs * proglucagon * brush border membrane sodium-dependent glucose transporter * basolateral glucose transporter * short chain fatty acids

The hypothesis that a conductive pathway for oxalate and chloride exists in brush-border membrane vesicle fractions prepared from a rabbit distal ileum was investigated. Results showed the presence of poorly saturated voltage-sensitive oxalate and chloride uptake pathways with low activation energies and their inhibition by several putative calcium channel blockers. These suggested that an apical membrane conductive pathway for the oxalate anion could contribute to cAMP-stimulated oxalate secretion.

Research was conducted to develop a technique to quantitate the contributions of NHE2 and NHE3 to rabbit ileal brush-border Na+/H+ exchange. Two novel methods were created which included the functional separation of the contribution of NHE2 and NHE3 to brush-border (BB) Na+/H+ exchange activity through the use of HOE-694 and quantitation of BBNHE3 through quantitative Western analysis. Results suggest that methylprednisolone stimulates ileal BB Na+/H+ exchange activity only by an effect on NHE3 and not on NHE2 and only by increasing the amount of BB NHE3.

Small intestinal brush-border hydrolases usually are assayed in intestinal mucosal homogenates resuspended in solutions of unphysiological ionic composition. Thus, extrapolation of measured [V.sub.max] values (maximal reaction rates at high substrate concentrations) to in vivo conditions, hence comparison with physiological substrate loads, is uncertain. We therefore have developed a sucrase assay in an intact preparation of mouse small intestine, an everted intestinal sleeve incubated in a physiological Ringer's solution. As in homogenate studies, sucrase is assayed by glucose production measured colorimetrically, but uptake of liberated glucose into the intestinal sleeve is prevented by the transport inhibitor phlorizin. The coefficient of variation of [V.sub.max] is 16% for sleeves from the same mouse and 8% for mean values from different mice. Sleeve sucrase activity is abolished by the inhibitor castanospermine. Activity in sleeves and homogenates proves to be the same when measured under identical solution conditions, but variations in assay conditions cause large activity changes from values measured in physiological solutions.

Safety factors of enzymes and transporters are defined as the ratio of [V.sub.max] (maximal reaction rates at high substrate concentrations) to the reaction rate under actual physiological conditions. Although corresponding safety factors have been measured for macroscopic biological structures and for human-engineered structures, safety factors have been little studied at the molecular level. Some evolutionary considerations suggest that safety factors should be modestly in excess of 1.0 ('enough but not too much') and should tend to be similar for the various steps of a pathway consisting of two or more elements arranged in series. Hence we used a preparation of intact mouse small intestine to measure [V.sub.max] values (capacities) of brush-border sucrase (yielding glucose plus fructose) and of the brush-border glucose transporter, for comparison with each other and with dietary sucrose loads. Load was manipulated by varying dietary sucrose level or by studying lactating mice with increased energy requirements. Capacities both of sucrase and the glucose transporter increased with sucrose load (i.e., both proteins are inducible) and remained approximately matched to each other except on a carbohydrate-free diet. Their safety factors decreased from ca. 2.7 at low load to 1.0 at high load. Thus, neither sucrase nor the glucose transporter is the rate-limiting step for sucrose digestion; both steps are equally limiting. The modest safety factors and matched capacities must be genetically programmed through natural selection, with benefits of excess capacities being balanced against costs of biosynthetic energy and limited membrane space.

Research demonstrates that brush border membrane marker enzymes gamma-glutamyltransferase and alkaline phosphatase during acute renal failure are proportionately less active in a progressively ischemic environment. Experiments designed to investigate the impact of ischemia on enzyme activity using young male Wistar rats described. Renal biochemical measurements taken over varying ischemia duration times are illustrated.

Research was conducted to study brush border myosin-I structure and adenosine diphosphate (ADP)-dependent conformational changes revealed by cryoelectron microscopy and image analysis. Three-dimensional maps of actin filaments decorated with BBM-I in both the presence and absence of 1 micron MgADP were generated using cryoelectron microscopy and helical image analysis. Comparison of the behavior of BBM-I with smooth and skeletal muscle subfragments-1 indicate significant differences in the energetics and structure of the biochemical transitions in the actomyosin ATPase cycle.

Research was conducted to study the transport of Na+-dependent D-glucose in the jejunal brush-border membrane vesicles (BBMV) of chickens from the posthatch period to 14 wk of age. Lipid-soluble fluorescent probe, 1,6-diphenyl-1,3-hexatriene was used to determine the steady-state fluorescence polarization and fluorescence anisotrophy. Results indicated that Na+-dependent D-glucose transport in jejunal BBMV decreased during the posthatching development. Other factors that may also influence transport are affinity of carrier proteins for the substrate and turnover of the transporter.

The monoclonal antibodies for high-resolution localization of NH3 in adult and neonatal rat kidney were examined employing immunofluorenscence and immunoelectron microscopy. Result of the study revealed that the nephron development, as shown by the pattern in NHE3 staining, was similar to that of an adult nephron. Furthermore, NHE3 is expressed on the apical membrane during early stages of development.

To better understand the effect of dietary fat on intestinal brush border (BB) membranes in the young animal, we compared the effect of dietary medium-chain triglycerides (MCT) with that of monounsaturated and saturated long-chain triglycerides (LCT) on jejunal brush border membrane lipid composition and hydrolase activity in newly weaned rats. Twenty-day-old rat pups were divided into three groups and were weaned to diets containing 14% MCT + 6% soybean oil, 18% olive oil + 2% soybean oil, or 14% tallow + 6% soybean oil, and fed for 40 h or for 33 d. The diets were isonitrogenous and contained similar amounts of cholesterol and polyunsaturated fatty acids. Within 40 h, the fatty acid compositions of the brush border membranes were significantly different among treatment groups. These differences were maintained in rats fed for 33 d. No medium-chain fatty acids, but significantly greater amounts of polyunsaturated fatty acids, especially 18:2(n-6) and 20:4(n-6), were found in the brush border membranes of rats fed the medium-chain triglyceride diet. The cholesterol and phospholipid concentrations in the membranes were highest in rats fed the medium-chain triglyceride diet for 33 d. Rats fed that diet for 40 h had generally higher leucine aminopeptidase, sucrase and maltase activities compared with rats fed the olive oil or tallow diets. However, after 33 d of feeding, the differences between dietary treatment groups disappeared. This study demonstrates that, in the newly weaned rat pup, dietary medium-chain triglycerides and long-chain triglycerides rapidly affect the fatty acid composition of the brush border membrane. However, the changes in the hydrolase activities associated with the changes in the lipid composition of the membranes are transient. J. Nutr. 126: 1455-1462, 1996. INDEXING KEY WORDS: * rats medium-chain triglycerides * long-chain triglycerides * membrane lipid * brush border hydrolases

Immunoblot analysis and immunocytochemistry techniques show that expression of myosin-IA (MIA) and myosin-IB (MIB) from Drosophila melanogaster is correlated with brush border cytoskeleton within the alimentary canal. In the larval gut, MIA is localized to the subapical terminal web domain whereas MIB is localized to the apical microvillar domain. In the adult gut, MIA also becomes predominant in the apical position in addition to its position in the web domain, while MIB attains a position in the egg chambers of the somatic follicle cells.

Transport of Na and glucose with a 1:1 stoichiometry and voltage-dependent kinetics in rainbow trout is done by a single Na-D-glucose cotransporter. Functions of the transporter are similar to mammalian transporters SGLT1 and SGLT2. The study is conducted using brush-border membrane vesicles, prepared from rainbow trout kidney and enriched with alkaline phosphatase and gamma-glutamyl transpeptidase.

A defect in the renal dopamine production in 12-mo-old rats results when there is a decreased uptake of dopa by tubular brush-border membrane. In 12-mo-old rats, the level of dopa and norepinephrine(NE) in the renal cortex are higher (P

Labeling of detergent-insoluble complexes for about 1 h shows the abundance of brush border enzymes in these complexes, which are isolated by density gradient centrifugation. There is an intracellular classification of the brush border membrane proteins to glycolipid 'rafts,' as revealed by the presence of alkaline phosphatase, aminopeptidase N and sucrase-isomaltase. The brush border enzymes target directly to the cell surface from the trans-Golgi network.

A study of the proneness of brush-border membrane hemileaflet to oxidative injury due to peroxyl radical using the peroxyl radical-generator AAPH and the lipid-soluble fluorescent probe cis-parinaric acid (PnA) reveals that the outer and inner hemileaflets exhibit different degradation patterns. Slow first-order kinetics characterize the degradation in the inner hemileaflet, while degradation in the outer hemileaflet is defined by two staggered first-order mechanism. The outer hemileaflet assimilates endogenous substrates for oxidation and other antioxidants faster than the inner hemileaflet.

A study of the regulation of proline transport across renal brush-border membrane (BBM) by Ca2+-dependent protein kinases reveals that NaCl-dependent proline transport across tubular luminal membrane is suppressed by Ca2+-depdendent, protein kinase-activated phosphorylation. The presence of Ca2+ calmodulin-dependent protein kinase II (CaMK II), and phospholipid- and Ca2+-dependent protein kinase C (PKC) in the BBM is established by bioassays using selective peptide substrates for PKC. Kinase inhibitor peptides reverse the proline suppression activated by CaMK and PKC.

Analysis of the level of differences in the fractional rates of disaccharide synthesis in brush-border membranes reveals that the sucrase synthesis in the membranes is low and this indicates the slow rate of processing of glycosylated precursor to the brush-border form. Stable isotopically labeled amino acids such as ((super 2)H(sub 3)) leucine are capable of determining the brush-border hydrolase and mucosal protein synthesis. The rates of protein synthesis in different mucosal compartment are different.

A comparative analysis of the binding activities of angiotensin II (ANG II) receptors in brush-border membranes (BBM) and basolateral membranes (BLM) of rabbit kidney reveals that the binding characteristics of BLM vary from that of BBM. The basal and luminal sides of the proximal tubules contain pharmacologically distinct ANG receptors, accounting for the different binding properties. The presence of a radiolabeled fragment affects the binding of ANG II receptors in BBM cells.

A new detergent-renaturation technique facilitates the purification of Bacillus thuringiensis CryIC in sodium dodecyl sulfate (SDS) which removes 10- and 25-kDa toxin segments that are bound to CryIC and modify membrane binding, along with suppressing iodination. The purified CryIC toxin is completely insecticidal to larvae of Spodoptera exigua. 125I-labeled CryIC attaches to S. exigua larvae brush border membrane vesicles with great affinity.

Diacyl and monoacyl phospholipids labeled with the fluorescent probe 7-nitrobenz-2-oxa-1,3-diazol-4-yl aid the estimation of the rate of phospholipid transport between artificial large unilammelar vesicles and brush-border membrane (BBM) vesicles of rabbit intestine. The intestinal brush border contains a protein transporter that is responsible for the absorption of short chain diacyl phospholipids and slightly water soluble lysophospholipids. Highly water insoluble long chain diacyl phospholipids are not redistributed between inner and outer leaflets by the BBM.

Studies on mice with X-linked hypophosphatemic Gy mutation suggest that the increased Na+-phosphate cotransport in the proximal tubule brush-border membrane is not the sole cause of enhanced renal phosphate conservation following phosphate deprivation. Consequently, the Gy mice possess reduced renal tubular maximum for phosphate.

A transmembrane Na gradient produced L-ascorbate transport by intestinal brush-border membrane vesicles in teleost Anguilla anguilla, while a potassium gradient produced no such effect. L-ascorbate transport was analyzed kinetically to express a nonsaturable transfer component with diffusional permeability, and single sodium influenced the carrier process. L-ascorbate influx was inhibited by D-Isoascorbate. On vitamin administration, an inside-negative membrane potential stimulated +Na-L-ascorbate, confirming its electrogenic character. L-ascorbate transport's Hill analysis revealed the sodium-ascorbate binding ratio to be 2:1.

Several peptidases found in the small intestinal brush-border membrane are involved in hydrolyzing dietary particles having protein. One of the many groups of rats under investigation were given a dosage of high-proline (gelatin) diet to determine the regulatory effects of the prolyl peptidases or the angiotensin-converting enzyme (ACE) and dipeptidyl peptidase IV(DPP IV) on the levels of hydrolysis. There was a three to sixfold increase in the DPP IV. The high-gelatin group exhibited a six to eight-fold increase. The results reveal that the increase in intestinal levels of the two enzymes is effective when there is a high-proline (gelatin).

Organic osmolyte betaine transport was observed and studied in rabbit renal brush-border membrane vesicles (BBMV). Na+ betaine and H+-betaine transport is electrogenic in character, as concentrative induction was observed only during occurrence of membrane potential. Chloride presence in the medium had no influence on betaine. L-protine antagonized Na+- and H+- betaine transport, and the H+- sensitive betaine pathway alone was inhibited by glycine. The highest rate of net betaine transport (Jmax) was enhanced by the simultaneous presence of Na+ and H+ gradients. Two unique betaine transport pathways in BBMV were identified. These pathways are defined by their Na+ or H+ sensitivity and their proline and glycine specificity.

Steady-state fluorescence polarization and differential phase fluorescence variations of 1,6-diphenyl-1,3,5-hexatriene (DPH) were used to study the effects of cholesterol oxidation by cholesterol oxidase on renal brush border membranes. Results revealed that DPH is distributed uniformly in outer and inner leaflets of the membrane and that its steady-state anisotropy decreases with cholesterol oxidation, a modification that resulted in a change in lipid order. Results support the existence of a weak interaction between other membrane lipids and the cholesterol readily accessible to cholesterol oxidase.

Two high molecular weight cytoskeletal proteins were identified as moieties of the intestinal epithelial cell brush borders. The larger of the twoprotein was designated as T-protein while the other was termed as N-protein. Immunofluorescence technique localized both proteins in the brush border terminal web near the microvilli. The molecular weights of T-protein and N-protein were noted to be similar to those of titin and nebulin respectively. T-protein was believed to be an isoform of titin and the results suggested structural roles of both proteins in the brush borders.

A study was conducted to prove that folate binding protein (FBP) from kidney brush border membranes contains a glycoinositol phospholipid anchor (GPL). Brush border membranes were isolated and labeled via (3H) folate. Results revealed the presence of individual residues common in GPL anchors which were covalently associated with FBP. Further tests also clarified the link between GPL-anchored FBP in biologocal membranes and soluble FBP in biological fluids.

The dynamics of polypeptide folding of pig intestinal enzymes was studied using a polyvalent antiserum for protein precipitation. The introduction of fructose produced a decrease in protein folding of the bruch border enzymes, aminopeptidase N and sucrase-isomaltase by interfering with glycosylation. Malglycosylated and high-mannose-glycosylatedforms are retained in the endoplasmic reticulum and degraded by proteolysis. The findings give importance to correct sequencing of glycosylation and polypeptide folding in intestinal brush border enzyme kinetics.

The binding of cholera toxin B subunit (CTB) to the brush border and its internalization are characterized. A marked shortening of the microvilli accompanied the toxin internalization whereas no formation of caveolae was observed.

In examining the functional aspects of human milk oligosaccharides (HMO), it is not known whether they are digested during the passage through the infant's gastrointestinal tract. HMO were prepared from individual milk samples (n = 6) and separated into neutral and acidic compounds by chromatography. These oligosaccharide fractions were studied for their digestibility by human salivary amylase, porcine pancreatic amylase and brush border membrane vesicles (BBMV) isolated from porcine small intestine; we also examined the effect of low pH on these structures. The characterization of HMO and their digestion products was performed by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) as well as TLC. It was shown that neither salivary amylase nor pancreatic amylase cleaved HMO. Only after a 2-h incubation with BBMV were slight modifications of the HMO observed. HPAEC-PAD analysis revealed two new components within the neutral oligosaccharide fractions; these were characterized by mass spectrometric analysis as lacto-N-triose and galactose. Only lacto-N-triose was present within digestion assays of oligosaccharides, which did not contain fucosyl or N-acetylneuraminic acid residues. These results suggest that [is less than] 5% of the HMO are digested in the intestinal tract. Hence, HMO may play a role as prebiotics or as factors influencing the local immune system of the intestine in breast-fed infants. J. Nutr. 130: 3014-3020, 2000. KEY WORDS: * milk oligosaccharides * digestion * humans * brush border membrane vesicles

Guo, P., A. M. Weinstein, and S. Weinbaum. A hydrodynamic mechanosensory hypothesis for brush border microvilli. Am J Physiol Renal Physiol 279: F698-F712, 2000.--In the proximal tubule of the kidney, [Na.sup.+] and [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] reabsorption vary proportionally with changes in axial flow rate. This feature is a critical component of glomerulotubular balance, but the basic mechanism by which the tubule epithelial cells sense axial flow remains unexplained. We propose that the microvilli, which constitute the brush border, are physically suitable to act as a mechanosensor of fluid flow. To examine this hypothesis quantitatively, we have developed an elastohydrodynamic model to predict the forces and torques along each microvillus and its resulting elastic bending deformation. This model indicates that: 1) the spacing of the microvilli is so dense that there is virtually no axial velocity within the brush border and that drag forces on the microvilli are at least 200 times greater than the shear force on the cell's apical membrane at the base of the microvilli; 2) of the total drag on a 2.5-[micro]m microvillus, 74% appears within 0.2 [micro]m from the tip; and 3) assuming that the structural strength of the microvillus derives from its axial actin filaments, then a luminal fluid flow of 30 nl/min produces a deflection of the microvillus tip which varies from about 1 to 5% of its 90-nm diameter, depending on the microvilli length. The microvilli thus appear as a set of stiff bristles, in a configuration in which changes in drag will produce maximal torque. glomerulotubular balance; mechanosensory mechanism; actin cytoskeleton; microvilli force and torque

Traebert, M., J. Roth, J. Biber, H. Murer, and B. Kaissling. Internalization of proximal tubular type II Na-[P.sub.i] cotransporter by PTH: immunogold electron microscopy. Am. J. Physiol. Renal Physiol. 278: F148-F154, 2000.--Physiological/pathophysiological alterations in proximal tubular [P.sub.i] reabsorption are associated with an altered brush-border membrane (BBM) expression of type II Na-[P.sub.i] cotransporter molecules. Reduction is achieved by an internalization and lysosomal degradation and an increase in [P.sub.i] reabsorption by new synthesis and BBM insertion of type II Na-[P.sub.i] cotransporters. In the present study, we investigated by immunohistochemistry and immunogold electron microscopy the routing of internalized rat type II Na-[P.sub.i] cotransporters (NaPi-2). In kidney of rats on a chronic low-[P.sub.i] diet, NaPi-2 is mainly localized in the BBM, in cisterns of the Golgi apparatus and sparsely also in large endocytotic vacuoles and lysosomes. Fifteen minutes after the injection of the 1-34 analog of parathyroid hormone (PTH), the amount of NaPi-2 was decreased in the BBM and increased in endocytotic vesicles. NaPi-2 molecules colocalized with horseradish peroxidase injected prior to the injection of PTH. Vesicles labeled for NaPi-2 were occasionally also labeled for clathrin or the adaptor protein AP2. We conclude that NaPi-2 molecules enter the subapical compartment from where NaPi-2-containing vesicles are segregated off and directed to the lysosomes. A clathrin-mediated pathway may contribute to the PTH-induced internalization of NaPi-2. NaPi-2; horseradish peroxidase; endocytosis; immunohistochemistry

Research was conducted to examine the role of villin, an actin-binding protein localized in intestinal and kidney brush borders, in vivo. The objective was to investigate the bundling and severing of actin microfilaments in the absence of villin. Results indicate that, in vivo, the bundling activity of villin is dispensable while the severing activity is not. It was demonstrated that villin is necessary for survival in experimental colitis resulting from the oral administration of dextran sulfate sodium.