AcMNPV P74 is cleaved at R325 and R334 by proteinases of both OB and BBMV to expose a potential fusion peptide for oral infection.

Baculoviruses enter insect midgut epithelial cells via a set of occlusion-derived virion (ODV) envelope proteins called per os infectivity factors (PIFs). P74 of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), which was the first identified PIF, is cleaved by an endogenous proteinase embedded within the occlusion body during per os infection, but the target site(s) and function of the cleavage have not yet been ascertained. Here, based on bioinformatics analyses, we report that cleavage was predicted at an arginine and lysine-rich region in the middle of P74. A series of recombinant viruses with site-directed mutants in this region of P74 were generated. R325 or R334 was identified as primary cleavage site. In addition, we showed that P74 is also cleaved by brush border membrane vesicles (BBMV) of the host insect at R325 or R334, instead of R195, R196, and R199, as previously reported. Simultaneous mutations in R195, R196, and R199 lead to instability of P74 during ODV release. Bioassays showed that mutations at both R325 and R334 significantly affected oral infectivity. Taken together, our data show that both R325 and R334 of AcMNPV P74 are the primary cleavage site for both occlusion body endogenous proteinase and BBMV proteinase during ODV release and are critical for oral infection. IMPORTANCE: Cleavage of viral envelope proteins is usually an important trigger for viral entry into host cells. Baculoviruses are insect-specific viruses that infect host insects via the oral route. P74, a per os infectivity factor of baculoviruses, is cleaved during viral entry. However, the function and precise cleavage sites of P74 remain unknown. In this study, we found that R325 or R334 between the N- and C-conserved domains of P74 was the primary cleavage site by proteinase either from the occlusion body or host midgut. The biological significance of cleavage seems to be the release of the potential fusion peptide at the N-terminus of the cleaved C-terminal P74. Our results shed light on the cleavage model of P74 and imply its role in membrane fusion in baculovirus per os infection.

Cinnamaldehyde improves the growth performance and digestion and absorption capacity in grass carp (Ctenopharyngodon idella).

The present study evaluated the effect of cinnamaldehyde (CIN) on the growth performance and digestion and absorption capacity of grass carp (Ctenopharyngodon idella). Fish were fed five diets including graded levels of CIN for 60 days. The results indicated that (1) appropriate CIN supplementation increased the growth performance and promoted the intestine growth of grass carp; (2) dietary appropriate CIN supplementation increased the digestion and absorption capacity by increasing the activities of intestinal and hepatopancreas digestive enzymes (lipase, chymotrypsin, trypsin, and amylase) and intestinal brush border enzymes (creatine kinase (CK), Na+/K+-ATPase, γ-glutamyl transpeptidase (γ-GT), and alkaline phosphatase (AKP)); (3) dietary CIN increased the absorption capacity which may be associated with the upregulated messenger RNA (mRNA) abundances of their amino acid transporters (AATs) in the intestine, which might be associated with activating the target of rapamycin (TOR) signaling pathway. The best CIN supplementation in the diets of grass carp was estimated to be 76.40 mg kg-1 diet based on the best percent weight gain (PWG). In general, CIN increased the digestion and absorption capacity of grass carp and raised the mRNA abundances of AATs which may be partly related to activation of the TOR signaling pathway.

Probiotic Pediococcus pentosaceus GS4 shields brush border membrane and alleviates liver toxicity imposed by chronic cadmium exposure in Swiss albino mice.

AIMS: To evaluate the protective efficacy of Pediococcus pentosaceus GS4 in cadmium (Cd)-induced toxicity in Swiss albino mice. METHODS AND RESULTS: The previously reported probiotic P. pentosaceus GS4 interaction with Cd was evaluated experimentally. Swiss albino mice were segregated randomly into three different groups containing nine animals each. The binding ability of P. pentosaceus GS4 and Cd was demonstrated by atomic absorption spectrometry, Fourier transform infrared spectroscopy and scanning electron microscopy analysis respectively. Cd challenge was attenuated by the P. pentosaceus GS4 treatment and was evaluated by estimating Cd level in liver and faeces, corresponding biomarkers for toxicity in liver and intestines and histopathological analysis with brush border membrane (BBM) integrity. Results showed that P. pentosaceus GS4 bound Cd effectively. Pediococcus pentosaceus GS4 intervention significantly enhanced faecal evacuation of Cd with an effect of reduced tissue deposition. Histologically, Cd-induced gut was found significantly replenished with lactobacilli in addition to reduced hyperplasia, lowered lymphocytes infiltration and enhancement of BBM-based disaccharidases, proving its role in protecting membrane integrity. CONCLUSIONS: Probiotic P. pentosaceus GS4 efficiently alleviated toxicity from vital organs such as liver and intestine caused by Cd exposure in a murine model. SIGNIFICANCE AND IMPACT OF THE STUDY: Intervention of P. pentosaceus GS4 alleviated Cd-induced toxicity, and thus it may be employed therapeutically to protect Cd toxicity.

Biochemical characterization of digestive membrane-associated alkaline phosphatase from the velvet bean caterpillar Anticarsia gemmatalis.

In Brazil, the use of transgenic plants expressing the insect-toxic Bacillus thuringiensis endotoxin has been successfully used as pest control management since 2013 in transgenic soybean lineages against pest caterpillars such as Helicoverpa armigera. These toxins, endogenously expressed by the plants or sprayed over the crops, are ingested by the insect and bind to receptors in the midgut of these animals, resulting in disruption of digestion and lower insect survival rates. Here, we identified and characterized a membrane-associated alkaline phosphatase (ALP) in the midgut of Anticarsia gemmatalis, the main soybean defoliator pest in Brazil, and data suggested that it binds to Cry1Ac toxin in vitro. Our data showed a peak of ALP activity in homogenate samples of the midgut dissected from the 4th and 5th instars larvae. The brush border membrane vesicles obtained from the midgut of these larvae were used to purify a 60 kDa ALP, as detected by in-gel activity and in vitro biochemical characterization using pharmacological inhibitors and mass spectrometry. When Cry1Ac toxin was supplied to the diet, it was efficient in decreasing larval weight gain and survival. Indeed, in vitro incubation of Cry1Ac toxin with the purified ALP resulted in a 43% decrease in ALP specific activity and enzyme-linked immunosorbent assay showed that ALP interacts with Cry1Ac toxin in vitro, thus suggesting that ALP could function as a Cry toxin ligand. This is a first report characterizing an ALP in A. gemmatalis.

Degradation of ß-casomorphin-7 through in vitro gastrointestinal and jejunal brush border membrane digestion.

This work aimed to study the opioid peptide ß-casomorphin-7 (BCM7) degradation or stability during digestion using human gastrointestinal (GI) juices and porcine jejunal brush border membrane (BBM) peptidases. Synthetic BCM7 was subjected to in vitro digestion by GI fluids obtained from human volunteers for 180 min, and to downstream degradation with porcine BBM vesicles. The BCM7 was sampled at 4 time points over 24 h after BBM addition. The digests were profiled by HPLC-electrospray ionization mass spectrometry (ESI/MS) to monitor BCM7 during GI digestion, and intact BCM7 through BBM digestion was quantified by reverse-phase (RP)-HPLC. We found that BCM7 was partly digested with human GI enzymes, as 3 proteolytic fragments in addition to f(60-66) YPFPGPI were detected: f(62-66) FPGPI, f(60-65) YPFPGP and f(61-66) PFPGPI. The RP-HPLC analysis revealed that 42% of the initial peptide was degraded after only 2 h of BBM digestion, and as much as 79% was degraded after 4-h digestion with supplementation of BBM. In conclusion, this study showed that most of BCM7 was degraded during GI and BBM digestion, although a small amount (5%) was still detected after 24-h digestion. It remains to be studied whether the small amount of intact BCM7 detected after in vitro digestion is transported via active transceptors in the human intestinal epithelial cells and enters blood circulation.

Cry6Aa1, a Bacillus thuringiensis nematocidal and insecticidal toxin, forms pores in planar lipid bilayers at extremely low concentrations and without the need of proteolytic processing.

Cry6Aa1 is a Bacillus thuringiensis (Bt) toxin active against nematodes and corn rootworm insects. Its 3D molecular structure, which has been recently elucidated, is unique among those known for other Bt toxins. Typical three-domain Bt toxins permeabilize receptor-free planar lipid bilayers (PLBs) by forming pores at doses in the 1-50 µg/ml range. Solubilization and proteolytic activation are necessary steps for PLB permeabilization. In contrast to other Bt toxins, Cry6Aa1 formed pores in receptor-free bilayers at doses as low as 200 pg/ml in a wide range of pH (5.5-9.5) and without the need of protease treatment. When Cry6Aa1 was preincubated with Western corn rootworm (WCRW) midgut juice or trypsin, 100 fg/ml of the toxin was sufficient to form pores in PLBs. The overall biophysical properties of the pores were similar for all three forms of the toxin (native, midgut juice- and trypsin-treated), with conductances ranging from 28 to 689 pS, except for their ionic selectivity, which was slightly cationic for the native and midgut juice-treated Cry6Aa1, whereas dual selectivity (to cations or anions) was observed for the pores formed by the trypsin-treated toxin. Enrichment of PLBs with WCRW midgut brush-border membrane material resulted in a 2000-fold reduction of the amount of native Cry6Aa1 required to form pores and affected the biophysical properties of both the native and trypsin-treated forms of the toxin. These results indicate that, although Cry6Aa1 forms pores, the molecular determinants of its mode of action are significantly different from those reported for other Bt toxins.

An Efficient Method for Labeling Single Domain Antibody Fragments with 18F Using Tetrazine- Trans-Cyclooctene Ligation and a Renal Brush Border Enzyme-Cleavable Linker.

Single domain antibody fragments (sdAbs) labeled with 18F have shown promise for assessing the status of oncological targets such as the human epidermal growth factor receptor 2 (HER2) by positron emission tomography (PET). Earlier, we evaluated two residualizing prosthetic agents for 18F-labeling of anti-HER2 sdAbs; however, these methods resulted in poor labeling yields and high uptake of 18F activity in the kidneys. To potentially mitigate these limitations, we have now developed an 18F labeling method that utilizes the trans-cyclooctene (TCO)-tetrazine (Tz)-based inverse-electron demand Diels-Alder reaction (IEDDAR) in tandem with a renal brush border enzyme-cleavable glycine-lysine (GK) linker in the prosthetic moiety. The HER2-targeted sdAb 2Rs15d was derivatized with TCO-GK-PEG4-NHS or TCO-PEG4-NHS, which lacks the cleavable linker. As an additional control, the non HER2-specific sdAb R3B23 was derivatized with TCO-GK-PEG4-NHS. The resultant sdAb conjugates were labeled with 18F by IEDDAR using [18F]AlF-NOTA-PEG4-methyltetrazine. As a positive control, the 2Rs15d sdAb was radioiodinated using the well-characterized residualizing prosthetic agent, N-succinimidyl 4-guanidinomethyl-3-[125I]iodobenzoate ([125I]SGMIB). Synthesis of [18F]AlF-NOTA-Tz-TCO-GK-2Rs15d was achieved with an overall radiochemical yield (RCY) of 17.8 ± 1.5% ( n = 5) in 90 min, a significant improvement over prior methods (3-4% in 2-3 h). In vitro assays indicated that [18F]AlF-NOTA-Tz-TCO-GK-2Rs15d bound with high affinity and immunoreactivity to HER2. In normal mice, when normalized to coinjected [125I]SGMIB-2Rs15d, the kidney uptake of [18F]AlF-NOTA-Tz-TCO-GK-2Rs15d was 15- and 28-fold lower ( P < 0.001) than that seen for the noncleavable control ([18F]AlF-NOTA-Tz-TCO-2Rs15d) at 1 and 3 h, respectively. Uptake of [18F]AlF-NOTA-Tz-TCO-GK-2Rs15d in HER2-expressing SKOV-3 ovarian carcinoma xenografts implanted in athymic mice was about 80% of that seen for coinjected [125I]SGMIB-2Rs15d. On the other hand, kidney uptake was 5-6-fold lower, and as a result, tumor-to-kidney ratios were 4-fold higher for [18F]AlF-NOTA-Tz-TCO-GK-2Rs15d than those for [125I]SGMIB-2Rs15d. SKOV-3 xenografts were clearly delineated even at 1 h after administration of [18F]AlF-NOTA-Tz-TCO-GK-2Rs15d by Micro-PET/CT imaging with even higher contrast observed thereafter. In conclusion, this strategy warrants further evaluation for labeling small proteins such as sdAbs because it offers the benefits of good radiochemical yields and enhanced tumor-to-normal tissue ratios, particularly in the kidney.

Acute renal toxicity of sodium chlorate: Redox imbalance, enhanced DNA damage, metabolic alterations and inhibition of brush border membrane enzymes in rats.

Sodium chlorate (NaClO3 ) is widely used in paper and pulp industries and as a non-selective herbicide. Humans can be exposed to NaClO3 through contaminated drinking water due to its improper and unchecked usage in industries and as herbicide. NaClO3 is also present as a major stable by-product in drinking water that has been disinfected with chlorine dioxide. In this study, we have investigated the effect of a single acute oral dose of NaClO3 on rat kidney. Adult male Wistar rats were divided into one control and four NaClO3 treated groups that were orally given different doses of NaClO3 and euthanized 24 hr after the treatment. Oral administration of NaClO3 resulted in increased hydrogen peroxide levels, lipid, and protein oxidation while thiol and glutathione content and activities of brush border membrane enzymes were decreased in kidney in a NaClO3 dose-dependent manner. Significant alterations in the activities of enzymes involved in carbohydrate metabolism and antioxidant defense were also observed. Administration of NaClO3 induced DNA fragmentation and increased DNA-protein cross-linking. Histological studies showed marked damage in kidney from NaClO3 treated animals. These results strongly suggest that NaClO3 induces nephrotoxicity via redox imbalance that results in DNA and membrane damage, metabolic alterations and brush border membrane enzyme dysfunction.

The lipid 5-phoshatase SHIP2 controls renal brush border ultrastructure and function by regulating the activation of ERM proteins.

The microvillus brush border on the renal proximal tubule epithelium allows the controlled reabsorption of solutes that are filtered through the glomerulus and thus participates in general body homeostasis. Here, using the lipid 5-phosphatase Ship2 global knockout mice, proximal tubule-specific Ship2 knockout mice, and a proximal tubule cell model in which SHIP2 is inactivated, we show that SHIP2 is a negative regulator of microvilli formation, thereby controlling solute reabsorption by the proximal tubule. We found increased PtdIns(4,5)P2 substrate and decreased PtdIns4P product when SHIP2 was inactivated, associated with hyperactivated ezrin/radixin/moesin proteins and increased Rho-GTP. Thus, inactivation of SHIP2 leads to increased microvilli formation and solute reabsorption by the renal proximal tubule. This may represent an innovative therapeutic target for renal Fanconi syndrome characterized by decreased reabsorption of solutes by this nephron segment.

The Secretion and Action of Brush Border Enzymes in the Mammalian Small Intestine.

Microvilli are conventionally regarded as an extension of the small intestinal absorptive surface, but they are also, as latterly discovered, a launching pad for brush border digestive enzymes. Recent work has demonstrated that motor elements of the microvillus cytoskeleton operate to displace the apical membrane toward the apex of the microvillus, where it vesiculates and is shed into the periapical space. Catalytically active brush border digestive enzymes remain incorporated within the membranes of these vesicles, which shifts the site of BB digestion from the surface of the enterocyte to the periapical space. This process enables nutrient hydrolysis to occur adjacent to the membrane in a pre-absorptive step. The characterization of BB digestive enzymes is influenced by the way in which these enzymes are anchored to the apical membranes of microvilli, their subsequent shedding in membrane vesicles, and their differing susceptibilities to cleavage from the component membranes. In addition, the presence of active intracellular components of these enzymes complicates their quantitative assay and the elucidation of their dynamics. This review summarizes the ontogeny and regulation of BB digestive enzymes and what is known of their kinetics and their action in the peripheral and axial regions of the small intestinal lumen.

Effect of increasing the proportion of dietary concentrate on gastrointestinal tract measurements and brush border enzyme activity in Holstein steers.

The aim of this study was to determine the time course for adaptation of the reticulo-rumen, omasum, abomasum, and small intestine in response to an abrupt increase in the proportion of grain in the diet. Adaptive responses include tissue and digesta mass, small intestinal length, and brush border enzyme activity in the duodenum, proximal jejunum, and ileum. Twenty-five Holstein steers (213 ± 23 kg; 5 to 7 mo of age) were blocked by body weight, and within block were randomly assigned to 1 of 5 treatments: the control diet (CTRL; 92% chopped grass hay and 8% mineral and vitamin supplement on a dry matter basis) or a moderate grain diet (MGD; 50% chopped grass hay, 42% rolled barley grain, and 8% mineral and vitamin supplement) that was fed for 3 (MGD3), 7 (MGD7), 14 (MGD14), or 21 d (MGD21). Dry matter intake was limited to 2.25% of body weight to ensure that changes in dry matter intake did not confound the results. On the last day of the dietary exposure, steers were slaughtered 2 h after feeding. Reticulo-rumen tissue mass and ruminal epithelium mass in the ventral sac of the rumen were not affected by the MGD. Wet reticulo-ruminal digesta mass decreased from CTRL to MGD7 and then increased, but reticulo-ruminal digesta dry matter mass did not differ between treatments. Omasal mass, omasal tissue mass, and omasum digesta mass decreased linearly with the number of days fed MGD, but abomasal tissue mass tended to increase linearly. Duodenal tissue mass tended to increase linearly, and ileal length increased linearly with the number of days fed MGD. Lactase activity in the proximal jejunum increased linearly and maltase activity in duodenum tended to increase linearly with days fed MGD. Aminopeptidase N activity in the proximal jejunum increased cubically with days fed MGD, and dipeptidylpeptidase IV activity in ileum tended to decrease from CTRL to MGD14 and then tended to increase. Adaptation to a diet with a greater proportion of concentrate involves changes in the mass and length of regions of the gastrointestinal tract and brush border enzyme activity. These changes take place gradually over at least 3 wk.

Sodium chlorate, a major water disinfection byproduct, alters brush border membrane enzymes, carbohydrate metabolism and impairs antioxidant system of Wistar rat intestine.

Sodium chlorate (NaClO3 ) is a widely used nonselective herbicide. It is also generated as a by-product during disinfection of drinking water by chlorine dioxide. The purpose of this study was to evaluate the effect of NaClO3 on rat intestine. Adult male rats were randomly divided into five groups: control and remaining four groups were administered orally different doses of NaClO3 and sacrificed 24 h after the treatment. The administration of NaClO3 produced acute oxidative stress in the intestine, which manifested in the form of markedly enhanced malondialdehyde levels and carbonyl content and lowered total sulfhydryl groups and glutathione levels. The activities of several brush border membrane (BBM) enzymes were greatly reduced as compared to control. There were alterations in the activities of various enzymes of carbohydrate metabolism and those involved in maintaining the antioxidant defense system. Histological studies support the biochemical results showing NaClO3 dose-dependent increase in tissue damage. Thus, the present study shows that oral administration of NaClO3 decreases the activities of BBM enzymes, induces oxidative stress, alters metabolic pathways, and impairs the antioxidant system of rat intestine. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1607-1616, 2017.

Milk diets influence doxorubicin-induced intestinal toxicity in piglets.

Chemotherapy-induced gastrointestinal (GI) toxicity is a common adverse effect of cancer treatment. We used preweaned piglets as models to test our hypothesis that the immunomodulatory and GI trophic effects of bovine colostrum would reduce the severity of GI complications associated with doxorubicin (DOX) treatment. Five-day-old pigs were administered DOX (1 × 100 mg/m(2)) or an equivalent volume of saline (SAL) and either fed formula (DOX-Form, n = 9, or SAL-Form, n = 7) or bovine colostrum (DOX-Colos, n = 9, or SAL-Colos, n = 7). Pigs were euthanized 5 days after initiation of chemotherapy to assess markers of small intestinal function and inflammation. All DOX-treated animals developed diarrhea, growth deficits, and leukopenia. However, the intestines of DOX-Colos pigs had lower intestinal permeability, longer intestinal villi with higher activities of brush border enzymes, and lower tissue IL-8 levels compared with DOX-Form (all P < 0.05). DOX-Form pigs, but not DOX-Colos pigs, had significantly higher plasma C-reactive protein, compared with SAL-Form. Plasma citrulline was not affected by DOX treatment or diet. Thus a single dose of DOX induces intestinal toxicity in preweaned pigs and may lead to a systemic inflammatory response. The toxicity is affected by type of enteral nutrition with more pronounced GI toxicity when formula is fed compared with bovine colostrum. The results indicate that bovine colostrum may be a beneficial supplementary diet for children subjected to chemotherapy and subsequent intestinal toxicity.

Minimal short-term effect of dietary 2'-fucosyllactose on bacterial colonisation, intestinal function and necrotising enterocolitis in preterm pigs.

Human milk decreases the risk of necrotising enterocolitis (NEC), a severe gastrointestinal disease that occurs in 5-10 % of preterm infants. The prebiotic and immune-modulatory effects of milk oligosaccharides may contribute to this protection. Preterm pigs were used to test whether infant formula enriched with α1,2-fucosyllactose (2'-FL, the most abundant oligosaccharide in human milk) would benefit gut microbial colonisation and NEC resistance after preterm birth. Caesarean-delivered preterm pigs were fed formula (Controls, n 17) or formula with 5 g/l 2'-FL (2'-FL, n 16) for 5 d; eight 2'-FL pigs (50 %) and twelve Controls (71 %) developed NEC, with no difference in lesion scores (P=0·35); 2'-FL pigs tended to have less anaerobic bacteria in caecal contents (P=0·22), but no difference in gut microbiota between groups were observed by fluorescence in situ hybridisation and 454 pyrosequencing. Abundant α1,2-fucose was detected in the intestine with no difference between groups, and intestinal structure (villus height, permeability) and digestive function (hexose absorption, brush border enzyme activities) were not affected by 2'-FL. Formula enrichment with 2'-FL does not affect gut microbiology, digestive function or NEC sensitivity in pigs within the first few days after preterm birth. Milk 2'-FL may not be critical in the immediate postnatal period of preterm neonates when gut colonisation and intestinal immunity are still immature.

Influence of time, storage temperature and freeze/thaw cycles on the activity of digestive enzymes from gilthead sea bream (Sparus aurata).

In this study, we tested the effects of long-term storage (2 years) at -20 °C and short-term storage (several hours) in ice and freeze/thaw cycles on the activities of pancreatic, gastric and intestinal (brush border and cytosolic) digestive enzymes in a teleost fish species. The results revealed a significant lose in activity of pancreatic (trypsin, chymotrypsin, total alkaline proteases and α-amylase) and intestinal cytosolic (leucine-alanine peptidase) enzymes between 140 and 270 days of storage at -20 °C, whereas in contrast, the activity of all the assayed brush border enzymes remained constant during the first 2 years of storage at -20 °C. During short-term storage conditions, the most stable enzymes assayed were those of the enterocytes of the brush border, which did not show any change in activity after being held for 5 h in ice. Five freezing and thawing cycles did not affect the activity of the intestinal brush border enzymes and the cytosolic ones, whereas the activity of trypsin, α-amylase and bile-salt-activated lipase was significantly affected by the number of freezing and thawing cycles. No changes in pepsin activity were found in samples exposed to 1 and 2 freezing and thawing cycles.

Mast cell regulation of Na-glutamine co-transporters B0AT1 in villus and SN2 in crypt cells during chronic intestinal inflammation.

BACKGROUND: In the chronically inflamed rabbit small intestine, brush border membrane (BBM) Na-glutamine co-transport is inhibited in villus cells (mediated by B0AT1), while it is stimulated in crypt cells (mediated by SN2/SNAT5). How mast cells, known to be enhanced in the chronically inflamed intestine, may regulate B0AT1 in villus and SN2/SNAT5 in crypt cell is unknown. Thus, the aim of the present study is to determine the regulation of B0AT1 and SN2/SNAT5 by mast cells during chronic enteritis. METHODS: Chronic intestinal inflammation was induced in male rabbits with intra-gastric inoculation of Eimeria magna oocytes. Rabbits with chronic inflammation were treated with ketotifen (10 mg/day) or saline (Placebo) for 2 days. Villus and crypts cells were isolated from the rabbit intestine using the Ca++ chelation technique. Na/K-ATPase activity was measured as Pi from cellular homogenate. BBM vesicles (BBMV) were prepared from villus and crypt cells and uptake studies were performed using rapid filtration technique with (3)H-Glutamine. Western blot analyses were done using B0AT1 and SN2 specific antibodies. RESULTS: In villus cells, Na-glutamine co-transport inhibition observed during inflammation was completely reversed by ketotifen, a mast cell stabilizer. In contrast, in crypt cells, Na-glutamine co-transport stimulation was reversed to normal levels by ketotifen. Kinetic studies demonstrated that ketotifen reversed the inhibition of B0AT1 in villus cells by restoring co-transporter numbers in the BBM, whereas the stimulation of SN2/SNAT5 in crypts cells was reversed secondary to restoration of affinity of the co-transporter. Western blot analysis showed that ketotifen restored immune-reactive levels of B0AT1 in villus cells, while SN2/SNAT5 levels from crypts cell remained unchanged. CONCLUSION: In the present study we demonstrate that mast cells likely function as a common upstream immune pathway regulator of the Na-dependent glutamine co-transporters, B0AT1 in villus cells and SN2 in crypts cells that are uniquely altered in the chronically inflamed small intestine.

ß-Casein(94-123)-derived peptides differently modulate production of mucins in intestinal goblet cells.

We recently reported the identification of a peptide from yoghurts with promising potential for intestinal health: the sequence (94-123) of bovine ß-casein. This peptide, composed of 30 amino acid residues, maintains intestinal homoeostasis through production of the secreted mucin MUC2 and of the transmembrane-associated mucin MUC4. Our study aimed to search for the minimal sequence responsible for the biological activity of ß-CN(94-123) by using several strategies based on (i) known bioactive peptides encrypted in ß-CN(94-123), (ii) in silico prediction of peptides reactivity and (iii) digestion of ß-CN(94-123) by enzymes of intestinal brush border membranes. The revealed sequences were tested in vitro on human intestinal mucus-producing HT29-MTX cells. We demonstrated that ß-CN(108-113) (an ACE-inhibitory peptide) and ß-CN(114-119) (an opioid peptide named neocasomorphin-6) up-regulated MUC4 expression whereas levels of the secreted mucins MUC2 and MUC5AC remained unchanged. The digestion of ß-CN(94-123) by intestinal enzymes showed that the peptides ß-CN(94-108) and ß-CN(117-123) were present throughout 1·5 to 3 h of digestion, respectively. These two peptides raised MUC5AC expression while ß-CN(117-123) also induced a decrease in the level of MUC2 mRNA and protein. In addition, this inhibitory effect was reproduced in airway epithelial cells. In conclusion, ß-CN(94-123) is a multifunctional molecule but only the sequence of 30 amino acids has a stimulating effect on the production of MUC2, a crucial factor of intestinal protection.

Lysophosphatidic acid stimulation of NHE3 exocytosis in polarized epithelial cells occurs with release from NHERF2 via ERK-PLC-PKCδ signaling.

The Na(+)/H(+) exchanger 3 (NHE3) is a brush border (BB) Na(+)/H(+) antiporter that accounts for the majority of physiologic small intestinal and renal Na(+) absorption. It is regulated physiologically and in disease via changes in endocytosis/exocytosis. Paradoxically, NHE3 is fixed to the microvillar (MV) actin cytoskeleton and has little basal mobility. This fixation requires NHE3 binding to the multi-PDZ domain scaffold proteins Na(+)/H(+) exchanger regulatory factor (NHERF)1 and NHERF2 and to ezrin. Coordinated release of NHE3 from the MV cytoskeleton has been demonstrated during both stimulation and inhibition of NHE3. However, the signaling molecules involved in coordinating NHE3 trafficking and cytoskeletal association have not been identified. This question was addressed by studying lysophosphatidic acid (LPA) stimulation of NHE3 in polarized renal proximal tubule opossum kidney (OK) cells that occurs via apical LPA5 receptors and is NHERF2 dependent and mediated by epidermal growth factor receptor (EGFR), Rho/Rho-associated kinase (ROCK), and ERK. NHE3 activity was determined by BCECF/fluorometry and NHE3 microvillar mobility by FRAP/confocal microscopy using NHE3-EGFP. Apical LPA (3 µM)/LPA5R stimulated NHE3 activity, increased NHE3 mobility, and decreased the NHE3/NHERF2 association. The LPA stimulation of NHE3 was also PKCδ dependent. PKCδ was necessary for LPA stimulation of NHE3 mobility and NHE3/NHERF2 association. Moreover, the LPA-induced translocation to the membrane of PKCδ was both ERK and phospholipase C dependent with ERK acting upstream of PLC. We conclude that LPA stimulation of NHE3 exocytosis includes a signaling pathway that regulates fixation of NHE3 to the MV cytoskeleton. This involves a signaling module consisting of ERK-PLC-PKCδ, which dynamically and reversibly releases NHE3 from NHERF2 to contribute to the changes in NHE3 MV mobility.

Regulation of proximal tubule vacuolar H(+)-ATPase by PKA and AMP-activated protein kinase.

The vacuolar H(+)-ATPase (V-ATPase) mediates ATP-driven H(+) transport across membranes. This pump is present at the apical membrane of kidney proximal tubule cells and intercalated cells. Defects in the V-ATPase and in proximal tubule function can cause renal tubular acidosis. We examined the role of protein kinase A (PKA) and AMP-activated protein kinase (AMPK) in the regulation of the V-ATPase in the proximal tubule as these two kinases coregulate the V-ATPase in the collecting duct. As the proximal tubule V-ATPases have different subunit compositions from other nephron segments, we postulated that V-ATPase regulation in the proximal tubule could differ from other kidney tubule segments. Immunofluorescence labeling of rat ex vivo kidney slices revealed that the V-ATPase was present in the proximal tubule both at the apical pole, colocalizing with the brush-border marker wheat germ agglutinin, and in the cytosol when slices were incubated in buffer alone. When slices were incubated with a cAMP analog and a phosphodiesterase inhibitor, the V-ATPase accumulated at the apical pole of S3 segment cells. These PKA activators also increased V-ATPase apical membrane expression as well as the rate of V-ATPase-dependent extracellular acidification in S3 cell monolayers relative to untreated cells. However, the AMPK activator AICAR decreased PKA-induced V-ATPase apical accumulation in proximal tubules of kidney slices and decreased V-ATPase activity in S3 cell monolayers. Our results suggest that in proximal tubule the V-ATPase subcellular localization and activity are acutely coregulated via PKA downstream of hormonal signals and via AMPK downstream of metabolic stress.

Effect of dietary lysine on growth, intestinal enzymes activities and antioxidant status of sub-adult grass carp (Ctenopharyngodon idella).

The dietary lysine requirement of sub-adult grass carp (460 ± 1.5 g) was assessed by feeding diets supplemented with grade levels of lysine (6.6, 8.5, 10.8, 12.9, 15.0 and 16.7 g kg(-1) diet) for 56 days. The test diets (28% CP) contained fish meal, casein and gelatin as sources of intact protein, supplemented with crystalline amino acids. Weight gain (WG), feed intake and feed efficiency were significantly improved with increasing levels of lysine up to 12.9 g kg(-1) diet and thereafter declined (P < 0.05). Quadratic regression analysis of WG at 95% maximum response indicated lysine requirement was 10.9 g kg(-1) diet. Activities of trypsin, chymotrypsin, lipase, Na(+), K(+)-ATPase and alkaline phosphatase in intestine, creatine kinase activity in proximal and mid-intestine responded similar to WG (P < 0.05). In addition, lipid and protein oxidation decreased with increasing levels of lysine up to certain values and increased thereafter (P < 0.05); the anti-hydroxyl radical capacity, dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione-S-transferase (GST) activities and glutathione content were increased with increasing dietary lysine levels up to certain values in the detected tissues, except for hepatopancreatic GST. Requirement estimated on the basis of malondialdehyde content in intestine and hepatopancreas was 10.6 and 9.53 g lysine kg(-1) diet, respectively.