Brush Border Membrane Enzymes Research Articles

Postprandial kinetics of digestive function in rainbow trout (Oncorhynchus mykiss): genes expression, enzymatic activity and blood biochemistry as a practical tool for nutritional studies

Postprandial kinetics of genes expression of gastric (chitinase, pepsinogen) and intestinal (alkaline phosphatase, maltase) digestive enzymes and nutrient transporters (peptide transporter 1, sodium-glucose transporter 1), Brush Border Membrane (BBM) enzymes activity (alkaline phosphatase, leucine aminopeptidase, maltase, saccharase) and blood biochemistry (triglycerides, cholesterol, protein, albumin, glucose, amino acids) through NMR spectroscopy, were investigated in rainbow trout (Oncorhynchus mykiss) fed a commercial aquafeed. For this purpose, fish were starved 72 h and digestive tract and blood were sampled before the meal and at 1.5, 3, 6, 9, 12, and 24 h after feeding (T0, T1.5, T3, T6, T9, T12 and T24). The postprandial kinetic showed that the expression of the genes involved in digestion and nutrient transport, the activity of BBM enzymes, and the presence of metabolites in blood were stimulated in different ways by the presence of feed in the digestive tract. The expression of most genes peaked 3 h after meal except gastric pepsinogen and maltase in distal intestine that peaked at T9 and T12, respectively. The activity of BBM enzymes were stimulated differently based on the intestine tract. The plasma proteins level increased from T1.5 until T9, while the other blood parameters unvariated during the postprandial period. This study supplied useful information about the physiological effects a single meal as a potential tool for planning nutritional studies involving the digestive functions.

292 Effect of Providing Supplemental Milk and Liquid Starter Diet for 4 and 11 Days, Respectively, Post-Weaning on Feed Intake, Growth and Intestinal Structure and Function of Newly Weaned Piglets

Abstract Reduced piglet feed intake, immediately post-weaning (PW), leads to disruption of the intestinal barrier, often resulting in health issues such as diarrhea. The objective was to evaluate the effect of providing supplemental milk and liquid starter diet for 4 and 11 days PW, respectively, on feed intake, growth, and intestinal structure and function of newly weaned piglets. Weaned piglets [n = 587 (Large White x Landrace) x Duroc; 28 days old] ) were divided into 59 pen groups each containing 9 to 10 same sex (entire male or female) piglets. The pen groups were blocked by sex and weaning weight and provided with ad libitum access to one of 5 dietary treatments: 1) Dry pelleted starter diet (control; CON); 2) CON + liquid milk replacer for 4 days (M4); 3) CON + liquid milk replacer for 11 days (M11); 4) CON + liquid starter diet for 4 days (S4) and 5) CON + liquid starter diet for 11 days (S11). Pen groups were weighed at weaning, days 11, 20 and 28 PW and feed disappearance (dry matter basis) was recorded on each weighing day. On day 7 PW, 10 piglets per treatment were euthanized to collect jejunal and ileal tissue samples. Villus height (VH), crypt depth and brush-border membrane (BBM) enzyme activity were determined from the tissue samples. Data were analysed with PROC MIXED (SAS-version 9.4, SAS Institute Inc.) with treatment as a fixed effect, block as a random effect, weaning weight as a co-variate and days PW as the repeated measure. Pen group was the experimental unit. From day 0 to 11 PW, M11 increased average daily gain (ADG) by 57% and average daily feed intake (ADFI) by 48% compared with CON (P < 0.05). M11 increased ADG by 54% and ADFI by 38% compared with S4 (P < 0.05) from day 0 to 11 PW. However, ADG and ADFI did not differ among treatment groups from day 11 to 28 PW. The treatments did not affect feed conversion ratio (P > 0.05) from day 0 to 28 PW. At day 7 PW, M11 piglets had 37% higher jejunal VH than CON piglets (P < 0.05) and S11 piglets had 28% higher ileal VH than S4 piglets (P < 0.05). The treatments did not affect crypt depth (P > 0.05). M11 piglets had up to 1.5-fold greater ileal sucrase activity compared with M4, S4 and S11 piglets (P < 0.05) and 1.8-fold greater ileal maltase activity compared with S4 piglets (P < 0.05). In conclusion, M11 and S11 reduced the negative effects of weaning, as they were associated with increased feed intake and growth in early PW. Offering feed in liquid form for longer than 4 days PW improved intestinal structure and increased BBM enzyme activity in newly weaned piglets.

Exploring gut microbiota in adult Atlantic salmon (Salmo salar L.): Associations with gut health and dietary prebiotics

BackgroundThe importance of the gut microbiota for physiological processes in mammals is well established, but the knowledge of their functional roles in fish is still limited. The aims of this study were to investigate associations between variation in taxonomical composition of the gut microbiota and gut health status in Atlantic salmon and to explore possible modulatory effects of dietary prebiotics in one net-pen farm in open water. The fish with initial mean body weight of around 240 g were fed diets based on the same basal composition, either without (Ref diet) or with (Test diet) yeast cell wall based-prebiotics, during the marine production phase from December to September the following year. Sampling was conducted at three sampling time points: January, April, and September, with average water temperature of 3.9 ℃, 3.4 ℃ and 9.6 ℃, respectively.ResultsAs the fish progressed towards September, growth, brush border membrane enzyme activities, and the expression in the gut of most of the observed genes involved in immune (e.g., il8, cd4a, myd88, il1b, gilt, tgfb, cd8b and cd3), barrier (e.g., zo1, occludin, ecad, claudin25b and claudin15), and metabolism increased significantly. Lipid accumulation in pyloric enterocytes decreased remarkably, suggesting improvement of gut health condition. The growth of the fish did not differ between dietary treatments. Further, dietary prebiotics affected the gut health only marginally regardless of duration of administration. Regarding gut microbiota composition, a decrease in alpha diversity (Observed species, Pielou and Shannon) over time was observed, which was significantly associated with an increase in the relative abundance of genus Mycoplasma and decrease in 32 different taxa in genus level including lactic acid bacteria (LAB), such as Lactobacillus, Leuconostoc, and Lactococcus. This indicates that developmental stage of Atlantic salmon is a determinant for microbial composition. Multivariate association analysis revealed that the relative abundance of Mycoplasma was positively correlated with gut barrier gene expression, negatively correlated with plasma glucose levels, and that its relative abundance slightly increased by exposure to prebiotics. Furthermore, certain LAB (e.g., Leuconostoc), belonging to the core microbiota, showed a negative development with time, and significant associations with plasma nutrients levels (e.g., triglyceride and cholesterol) and gene expression related to gut immune and barrier function.ConclusionsAs Atlantic salmon grew older under large-scale, commercial farm settings, the Mycoplasma became more prominent with a concomitant decline in LAB. Mycoplasma abundance correlated positively with time and gut barrier genes, while LAB abundance negatively correlated to time. Dietary prebiotics affected gut health status only marginally.

Repetitive and zonal expression profiles of absorption-related genes in the gastrointestinal tract of ascidian Ciona intestinalis type A.

Intestinal absorption is essential for heterotrophic bilaterians with a tubular gut. Although the fundamental features of the digestive system were shared among chordates with evolution, the gut morphologies of vertebrates diverged and adapted to different food habitats. The ascidian Ciona intestinalis type A, a genome-wide research model of basal chordates, is used to examine the functional morphology of the intestines because of its transparent juvenile body. In the present study, the characteristic gene expression patterns (GEP) of Ciona absorptive proteins, e.g., brush border membrane enzymes for terminal digestion (lactase, maltase, APA, and APN) and transporters (SGLT1, GLUT5, PEPT1, and B0AT1), were investigated in juveniles and young adults, with a special reference to the absorption of other nutrients by pinocytosis- and phagocytosis-related proteins (megalin, cubilin, amnionless, Dab2, Rab7, LAMP, cathepsins, and MRC1). Whole-mount in situ hybridization revealed that these GEP showed multi-regional and repetitive features along the Ciona gastrointestinal tract, mainly in the stomach and several regions of the intestines. In young adults, many absorption-related genes, including pinocytosis-/phagocytosis-related genes, were also expressed between the stomach and mid-intestine. In the gastrointestinal epithelium, absorption-related genes showed zonal GEP along the epithelial structure. Comparisons of GEP, including other intestinal functions, such as nutrient digestion and intestinal protection, indicated the repetitive assignment of a well-coordinated set of intestinal GEP in the Ciona gastrointestinal tract.

Regulation of survival, growth performance, and intestinal health of large yellow croaker (Larimichthys crocea) larvae by sodium decanoate supplementation

A 30-day feeding trial was undertaken to elucidate the impact of diets supplemented with sodium decanoate (Sd) on survival, growth performance, and intestinal health in Larimichthys crocea larvae. A series of experimental diets consisting of graduated levels of Sd were formulated, including 0.0% (the control), 0.1%, 0.2%, 0.4%, or 0.8%. The study revealed no significant variations in survival among dietary groups. The growth performance (final body weight and specific growth rate) exhibited significant linear and quadratic trends, with the highest values observed in larvae fed a diet containing 0.2% Sd, which was significantly higher than that of the control group. Notably, Larvae fed diets with 0.2%–0.8% Sd had significantly higher intestinal fold height than the control group. A finding that was substantiated by the increased mRNA expression of pcna and odc, which are known to reflect processes of epithelial proliferation and differentiation. Meanwhile, the significant increase in the activity of the intestinal brush border membrane enzyme (Leucine-aminopeptidase), observed in diets supplemented with 0.1%–0.8% Sd, provides compelling evidence supporting the promotion of intestinal development and maturation. In terms of digestive and absorptive capacity, dietary supplementation with 0.4% Sd significantly increased the activity of trypsin and creatine kinase, while 0.1%–0.4% Sd specifically enhanced the activity of Na+-K+-Adenosinetriphosphatase than the control group. Importantly, larvae consuming Sd-supplemented diets at appropriate levels exhibited increased mRNA expression of tight junction-associated genes. Additionally, diets supplemented appropriate Sd improved the antioxidant capacity by modulating antioxidant enzymes and genes, antioxidant substances, and lipid peroxidation products. In summary, the diet incorporating 0.2% Sd could potentially boost the growth of larvae, which may be connected to Sd promoting intestinal health by regulating intestinal morphology and development, digestion and absorption capacity, intestinal barrier function, and antioxidant capacity. This study sheds light into the application of medium-chain fatty acids in marine fish larvae and contributes to the development of nutritional regulation strategies aimed at enhancing larval development.

Reduction of the Renal Radioactivity of 111In-DOTA-Labeled Antibody Fragments with a Linkage Cleaved by the Renal Brush Border Membrane Enzymes.

The interposition of a cleavable linkage by enzymes on the renal brush border membrane constitutes a promising approach for reducing the renal radioactivity levels of radiolabeled low-molecular-weight antibody fragments and constructs (LMW Abs). Herein, we applied the molecular design to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-based reagents for radiotheranostic applications with trivalent radiometals. DOTA or a derivative thereof was conjugated to a Fab through an FGK linkage ([111In]In-DO3AiBu-Bn-FGK-Fab or [111In]In-DOTA-Bn-FGK-Fab). When injected into mice, both generated radiometabolites, [111In]In-DO3AiBu-Bn-F and [111In]In-DOTA-Bn-F, by the angiotensin-converting enzyme at similar rates. Both exhibited significantly lower renal radioactivity levels than a 111In-labeled Fab prepared by the conventional procedure ([111In]In-DOTA-Bn-SCN-Fab). The different elimination rates of each radiometabolite from the kidney significantly affected the renal radioactivity levels. [111In]In-DO3AiBu-Bn-FGK-Fab preferentially reduced the renal localization without impairing tumor accumulation. These findings would pave the way for developing a DOTA-based radiotheranostic platform for LMW Abs bearing cleavable linkers for renal brush border enzymes.

Oral administration of Nigella sativa oil attenuates arsenic-induced redox imbalance, DNA damage, metabolic distress, and histopathological alterations in rat intestine

BackgroundExposure to arsenic, a widespread environmental toxin, produces multiple organ toxicity, including gastrointestinal toxicity. Nigella sativa (NS) has long been revered for its numerous health benefits under normal and pathological states. In view of this, the present study attempts to evaluate the protective efficacy of orally administered Nigella sativa oil (NSO) against arsenic-induced cytotoxic and genotoxic alterations in rat intestine and elucidate the underlying mechanism of its action. MethodsRats were categorized into the control, NaAs, NSO, and NaAs+NSO groups. After pre-treatment of rats in the NaAs+NSO and NSO groups daily with NSO (2 ml/kg bwt, orally) for 14 days, NSO treatment was further continued for 30 days, with and without NaAs treatment (5 mg/kg bwt, orally), respectively. Various biochemical parameters, such as enzymatic and non-enzymatic antioxidants, carbohydrate metabolic and brush border membrane marker enzyme activities were evaluated in the mucosal homogenates of all the groups. Intestinal brush border membrane vesicles (BBMV) were isolated, and the activities of membrane marker enzyme viz. ALP, GGTase, LAP, and sucrase were determined. Further, the effect on kinetic parameters viz KM (Michaelis-Menten constant) and Vmax of these enzymes was assessed. Integrity of enterocyte DNA was examined using the comet assay. Histopathology of the intestines was performed to evaluate the histoarchitectural alterations induced by chronic arsenic exposure and/or NSO supplementation. Arsenic accumulation in the intestine was studied by inductively coupled plasma-mass spectroscopy (ICP-MS). ResultsNaAs treatment caused substantial changes in the activities of brush border membrane (BBM), carbohydrate metabolism, and antioxidant defense enzymes in the intestinal mucosal homogenates. The isolated BBM vesicles (BBMV) also showed marked suppression in the marker enzyme activities. Severe DNA damage and mucosal arsenic accumulation were observed in rats treated with NaAs alone. In contrast, oral NSO supplementation significantly alleviated all the adverse alterations induced by NaAs treatment. Histopathological examination supported the biochemical findings. ConclusionNSO, by improving the antioxidant status and energy metabolism, could significantly alter the ability of the intestine to protect against free radical-mediated arsenic toxicity in intestine. Thus, NSO may have an excellent scope in managing gastrointestinal distress in arsenic intoxication.

Oral administration of curcumin and gallic acid alleviates pentachlorophenol-induced oxidative damage in rat intestine.

Pentachlorophenol (PCP) is used as pesticide and wood preservative. We have previously shown that PCP causes oxidative damage in rat intestine. This study aimed to delineate the possible therapeutic potential of curcumin (CUR) and gallic acid (GA) against PCP-induced damage in rat intestine. PCP alone group received 125mg PCP/kg body weight/day orally for 4 days. Animals in combination groups received CUR or GA (100mg/kg bw) for 18 days and PCP (125mg/kg bw) for the last four days. Rats were sacrificed and intestinal preparations were analyzed for various parameters. Administration of PCP alone altered the activities of metabolic, antioxidant and brush border membrane enzymes. It also increased DNA-protein crosslinking and DNA-strand scission. Animals in combinations groups showed significant amelioration against PCP-induced oxidative damage. Histological abrasions were seen in PCP alone group which were reduced in the intestines of combination groups. CUR was more effective protectant than GA. CUR and GA protected rat intestine from PCP-mediated changes in the activities of metabolic, antioxidant and brush border membrane enzymes. They also prevented DNA damage and histological abrasions. The antioxidant character of CUR and GA may be responsible for the diminution of PCP-mediated oxidative damage.

Effect of feed restriction and refeeding on body condition, digestive functionality and intestinal microbiota in rainbow trout (Oncorhynchus mykiss)

The aim of the present work was to investigate the influence of fasting and refeeding on body condition, gut physiology and microbiota in reared O. mykiss. Ninety-six fish were randomly allotted among three groups subjected to different feeding plan: C (control, fed for 5 weeks); R (restricted ration over 3 weeks followed by 2 weeks feeding); F (fasted over 3 weeks followed by 2 weeks feeding) in a well’s fresh water flow-through rearing plan. Sampling occurred at 0, 1, 2, 4, 7, 14 days during the refeeding period. At day 0 and throughout the feeding period until day 14, the weight of the fish was significantly affected by the feeding restriction. Feed deprivation reduced significantly the viscerosomatic and hepatosomatic indexes. Brush border membrane enzymes’ specific activity was modulated by feeding regimes until day 7, to level in all experimental groups at day 14. At the end of the restricted/fasted period, the microbiota of the C group was made up of 70% of Actinobacteria, 24% of Proteobacteria, 4.2% of Firmicutes and < 1% of Bacteroides, while the restricted and fasted group were characterized by a strong reduction of Actinobacteria, and a significant increase in Bacteroidetes and Firmicutes. The feed deprivation determined a dysbiosis, allowing the development of different commensal or pathogenic bacteria. In conclusion, the effects of 2 weeks of feed deprivation, excluding those related to body weight, are gradually mitigated by refeeding, which allows the restoration of digestive functions and a healthy intestinal microbiota.

Pentachlorophenol causes redox imbalance, inhibition of brush border membrane and metabolic enzymes, DNA damage and histological alterations in rat kidney

Pentachlorophenol (PCP) is a synthetic organochlorine compound that is widely used in biocide and pesticide industries, and in preservation of wood, fence posts, cross arms and power line poles. Humans are usually exposed to PCP through air, contaminated water and food. PCP enters the body and adversely affects liver, gastrointestinal tract, kidney and lungs. PCP is a highly toxic class 2B or probable human carcinogen that produces large amount of reactive oxygen species (ROS) within cells. This work aimed to determine PCP-induced oxidative damage in rat kidney. Adult rats were given PCP (25, 50, 100, 150 mg/kg body weight), in corn oil, once a day for 5 days while control rats were given similar amount of corn oil by oral gavage. PCP increased hydrogen peroxide level and oxidation of thiols, proteins and lipids. The antioxidant status of kidney cells was compromised in PCP treated rats while enzymes of brush border membrane (BBM) and carbohydrate metabolism were inhibited. Plasma level of creatinine and urea was also increased. Administration of PCP increased DNA fragmentation, cross-linking of DNA to proteins and DNA strand scission in kidney. Histological studies supported biochemical findings and showed significant damage in the kidneys of PCP-treated rats. These changes could be due to redox imbalance or direct chemical modification by PCP or its metabolites. These results signify that PCP-induced oxidative stress causes nephrotoxicity, dysfunction of BBM enzymes and DNA damage.

Fucoidan Improves Growth, Digestive Tract Maturation, and Gut Microbiota in Large Yellow Croaker (Larimichthys crocea) Larvae.

The early life period is considered an essential period for gut microbial colonization. Manipulating gut microbiota interventions during early life periods has been proven to be a promising method to boost healthy growth. Therefore, the aim of the present study was to investigate the effects of dietary fucoidan (Fuc) on the growth, digestive tract maturation, and gut microbiota of large yellow croaker (Larimichthys crocea) larvae. Four diets were formulated with different levels of Fuc (0.00%, 0.50%, 1.00%, and 2.00%). Results showed that dietary Fuc significantly improved the growth performance of larvae. Meanwhile, dietary Fuc promoted digestive tract maturation. Dietary 1.00% Fuc significantly improved intestinal morphology. Dietary Fuc upregulated the expression of intestinal cell proliferation and differentiation related-genes and intestinal barrier related-genes. Dietary 2.00% Fuc significantly increased the activities of brush border membranes enzymes and lipase while inhibiting α-amylase. Furthermore, dietary Fuc maintained healthy intestinal micro-ecology. In detail, dietary 1.00% and 2.00% Fuc altered the overall structure of the gut microbiota and increased the relative abundance of Bacteroidetes while decreasing the relative abundance of opportunistic pathogens and facultative anaerobe. In conclusion, appropriate dietary Fuc (1.00-2.00%) could improve the growth of large yellow croaker larvae by promoting digestive tract maturation and maintaining an ideal intestinal micro-ecology.

The beneficial effects of exogenous protease K originated from Parengyodontium album on growth performance of grass carp (Ctenopharyngodon idella) in relation to the enhanced intestinal digestion and absorption capacities

The study was primarily designed to analyze the effect of exogenous protease K on growth performance, amino acid metabolism, digestion and absorption capacities, expression of amino acid transporters related genes and proteins in intestine of sub-adult grass carp (Ctenopharyngodon idella). Six experimental diets with protease K (0, 3000, 6000, 9000, 12,000 and 15,000 U/kg) were fed to 450 grass carp (619.51 ± 0.54 g) for 60 days. The results indicated that the highest value of final body weight, percent weight gain (PWG), specific growth rate, protein efficiency ratio, activity of hepatopancreas glutamate-oxaloacetate transaminase and glutamate-pyruvate transaminase, and the lowest concentration of plasma ammonia were observed in groups with protease K of 6000 and 9000 U/kg. Meanwhile, the groups with 6000 and 9000 U/kg protease K exhibited the highest activities of digestive and brush border membrane enzymes in the three intestinal segments, as well as the highest value of intestinal somatic index and intestinal length index. The highest concentration of free amino acid in the serum and the lowest in intestine were observed in groups with supplementation of protease K 6000 and 9000 U/kg, which might be related to the up-regulated mRNA level and protein expression of intestinal amino acid transporters (SLC1A5, SLC7A6, SLC1A3 et al) in these groups. Furthermore, optimal levels of protease K up-regulated the mRNA levels of target of rapamycin (TOR), S6 kinase 1 (S6K1), and protein expression of TOR and phosphorylation TORSer2448, but reduced the mRNA level of 4E binding protein 1 (4EBP1). In summary, optimal levels of exogenous protease K was found to be conducive to facilitate the improvement of growth performance probably via enhancing digestion and absorption capacity, and utilization of amino acid in sub-adult grass carp. In addition, using quadratic regression analysis of PWG and alkaline phosphatase in the midgut, the optimum addition of protease K for the sub-adult grass carp were noted to be 7380.82 and 7406.44 U/kg, respectively.

Oral Administration of Copper Chloride Damages DNA, Lowers Antioxidant Defense, Alters Metabolic Status, and Inhibits Membrane Bound Enzymes in Rat Kidney.

Copper (Cu) is a heavy metal that is widely used in industries and is also an essential micronutrient for living beings. However, excess Cu is toxic and human exposure to high levels of this metal results in numerous adverse health effects. We have investigated the effect of oral administration of copper chloride (CuCl2), a Cu(II) compound, on various parameters of oxidative stress, cellular metabolism, and DNA integrity in the rat kidney. This was done to delineate the molecular mechanism of Cu(II) toxicity. Adult male rats were randomly divided into five groups. Animals in four CuCl2-treated groups were separately administered single acute oral dose of CuCl2 at 5, 15, 30, and 40mg/kg body weight. Animals in the fifth group were not given CuCl2 and served as the control. All rats were sacrificed 24h after the dose of CuCl2 and their kidneys removed. CuCl2 administration led to significant alterations in enzymatic and non-enzymatic parameters of oxidative stress. It changed the activities of metabolic and membrane bound enzymes and also decreased the activities of brush border membrane enzymes. CuCl2 treatment dose-dependently enhanced DNA damage and DNA-protein crosslinking in renal cells, when compared to the control group. The administration of CuCl2 also resulted in marked morphological changes in the kidney, with more prominent alterations at higher doses of CuCl2. These results clearly show that CuCl2 impairs the antioxidant defense system resulting in oxidative damage to the kidney.

Dietary Tryptophan‐Lactobacillus rhamnosus GG Interactions Modify Intestinal Differentiation and Function

Although the influence of the gut microbiota on structure and proliferation of intestinal epithelial cells has been established, there is disconcordance on information related to cell type differentiation, because most studies on probiotics reflected an outcome from interactions among Lactobacillus, numerous existing microbes, and the host, with limited insight on the specific impact of Lactobacilli spp. For example, L. reuteri enhanced goblet and Paneth cell differentiation in intestine of conventionally‐raised chickens with an established commensal gut microbiota, confounding results. Here we fed gnotobiotic mice with tryptophan (t+) or trp‐free (t–) diets, followed by monocolonization with LGG (L+, ATCC 53103) or without (control, L–) for a total of 4 groups (L+t+, L+t–, L–t+, L–,t–), then ileum transcriptome was analyzed 3 wk later. L– mice remained germ‐free throughout while only LGG was associated with L+. Principal component analysis shows L+ or t+ by itself to modestly cluster away from L–t–, but the cluster depicting the combination of L+t+ was far removed from the others. The heatmap of whole ileum transcriptome shows several distinct groups of genes affected by t+ or by L+ alone. However, the simultaneous impact of L+t+ dramatically upregulates numerous gene clusters while downregulating a few. Volcano plots and Venn diagrams comparing L–t– with L–t+, or L+t– with L–t– indicate ~2300 unique genes affected by L+ (LGG) or t+ (tryptophan) alone. Comparisons between L+t+ and L+t– (effect of t with L) or between L+t+ and L–t+ (effect of L with t) each impact &gt;3800 genes, suggesting that synergistic interactions between L+ and t+ primarily underlie the probiotic impact ascribed to LGG and dietary t. Many of the genes upregulated by L+t+ have functions typical of mature enterocytes: tight junctions, brush border membrane transporters and enzymes, intestinal bile acid metabolism, lipid synthesis and fatty acid b‐oxidation. Many downregulated genes are antimicrobial and secreted peptides typical of differentiated Paneth and goblet cells. This latter result was recapitulated when reexamined in conventional mice perfused for 4 h with LGG, as expression of many defensins decreased by &gt;10‐fold, suggesting not only that this probiotic’s effects can arise from both acute ex vivo and chronic in vivo interactions with the host, but also in hosts housing a complex microbiota. Remarkably, Notch ligands Dll1 and Dll4 typically expressed in Paneth cells as well as Yap1, Axin2 and Ctnnb1 are upregulated in L+t+ ileum, suggesting downstream upregulation of Hes1 and repression of Atoh1, leading to suppression of the secretory lineage. In conclusion, LGG monocolonization may lead to synthesis of t+‐derived metabolites by both host and bacteria, leading to an enhancement of absorptive enterocyte functions and suppression of those by goblet and Paneth cells.

Opportunities Lost? Evolutionary Causes and Ecological Consequences of the Absence of Trehalose Digestion in Birds.

AbstractTrehalose is a nonreducing disaccharide that is a primary storage and energy source in prokaryotes, yeasts, fungi, and invertebrates. Vertebrates digest trehalose with the intestinal brush border membrane (BBM) enzyme trehalase. Intestinal trehalase activity is reported to be either very low or absent in several bird species. We assayed trehalase activity in 19 avian species, used proteomic analysis to quantify its abundance in the intestinal BBM, and used analyses of available genomes to detect the presence of the gene that codes for trehalase (Treh). We found no intestinal trehalase activity in birds, trehalase was absent from the proteome of their intestinal BBM, and the gene coding for trehalase was absent in their genomes. Surveys of available transcriptomes support the hypothesis that Treh is absent in birds. The trehalase gene was found in the same conserved syntenic block within the genome of all vertebrates surveyed except birds. Our analysis suggests that Treh was lost in an inversion followed by a reinsertion of a large gene block. This event appears to have taken place after the split between crocodiles and birds and dinosaurs. Birds are unable to digest a common dietary sugar like trehalose because their ancestor lost the trehalase gene. The loss of this gene seems to represent an ecological cost, as insectivorous birds seem to be unable to digest a carbohydrate present in their prey. We also speculate that the paucity of mycophagy in birds is due to the presence of large amounts of this sugar in fungal tissues.

Effects of First Feeding Regime on Gene Expression and Enzyme Activity in Pikeperch (Sander lucioperca) Larvae

The present study investigates the effects of different feeding regimes with rotifers (Brachionus plicatilis) and Artemia salina on the gene expression and digestive enzymes in pikeperch (Sander lucioperca) larvae at 17 days post-hatch (DPH) over a period of 13 days. Five experimental feeding protocols were performed in four replicates. At 4 DPH, the larvae (total length= 5.62 ± 0.03 mm, body weight = 0.66 ± 0.16 mg) were divided into five experimental groups (2-L tanks) at initial density of 100 larvae per liter. Light intensity on the water surface was 90-100 lux and photoperiod was set at 13L: 11D (07:00 to 20:00 h). Water temperature, pH, and dissolved oxygen (DO) were measured before each feeding and the values were 17.8 ± 0.17°C, 7.3 ± 0.04 and 88.5 ± 2.53%. The fish larvae at 5 days post-hatch (DPH), were initially fed with rotifers (Brachionus plicatilis) for 3 days and from 8 to 17 DPH were fed with rotifers/Artemia for different time periods as follows: (A) only rotifers; (B) 8–13 DPH rotifers/14–17 DPH Artemia; (C) 8–10 DPH rotifers/11–17 DPH Artemia; (D) only Artemia; (E) a combination of rotifers and Artemia. Frozen paste of algae was added to the larval tanks twice a day (2 x 300,000 cells/mL). Rotifers and Artemia were provided as live feed to larvae three times a day with residual counts prior to each feeding. Feeding densities were steadily increased based on residual counts, performed prior to each feeding. The expression of genes related to intestinal development and maturation (aminopeptidase N, anpep; leucine aminopeptidase 3, lap3; intestinal-type alkaline phosphatase, alpi), together with key pancreatic digestive proenzymes (trypsinogen 1, try1; chymotrypsinogen b, ctrb; carboxyl ester lipase precursor, cel; phospholipase a2, pla2g1b; pancreatic alpha amylase, amy2a), were assessed. Additionally, the activity of six enzymes (trypsin, lipase, alkaline phosphatase, amino peptidase, amylase, and chymotrypsin) were determined. The highest expression of two genes related to intestine (lap3; anpep) were observed in the fish fed a combination of rotifers and Artemia from 8 DPH (Group E). The expression of amy2a, ctrb, pla2g1b, try1 was significantly lower in larvae fed rotifers until 14 DPH and replaced by Artemia afterwards (Group B). The specific activity of brush border membrane enzymes (alkaline phosphatase and aminopeptidase N) increased with combination of rotifers and Artemia in larval diet (Group E), indicating a more efficient functionality of digestive structures. The groups fed only with rotifers till 17 DPH (Group A) (38 ± 4.07%) and larvae fed with rotifers till 14 DPH followed by feeding with Artemia till 17 DPH (Group B) (36 ± 5.25%) showed significantly (P&amp;lt;0.05) lower survival rates than the other groups (54-67%). The group fed only with rotifers (Group A) showed significantly lower specific growth rate (SGR) than the other groups, and the highest SGR was found in the group fed with combination of rotifers and Artemia after 3 day rotifer feeding (Group E). The highest standard length (8.32 ± 0.48 mm) was obtained by combined feeding of rotifers and Artemia after 3 day of initial rotifer feeding. Combination of rotifers and Artemia from 8 DPH (Group E) could be considered a more appropriate diet for first feeding pikeperch larvae compared with later introduction of Artemia, as indicated by the higher expression of genes and activities of digestive enzymes. Our findings provide new insight into the effect of temporal sequence of rotifers and Artemia on the expression of genes and activities of digestive enzymes in pikeperch larvae.

Effect of sprouting on the proteome of chickpea flour and on its digestibility by ex vivo gastro-duodenal digestion complemented with jejunal brush border membrane enzymes

The demand for sustainably produced proteins is increasing with the world population and is prompting a dietary shift toward plant sourced proteins. Vegetable proteins have lower digestibility and biological value compared to animal derived counterparts. We explored sprouting of chickpea seeds as a strategy for improving digestibility. Protein evolution associated with by the sprouting process was assessed by proteomics. The sprouting induced breakdown of seed storage proteins and doubled the release of free alpha-amino nitrogen in sprouted chickpea flour. During sprouting, several enzymes involved in plant development were newly expressed. An ex vivo model of gastroduodenal and jejunal digestion was applied to assess the bioaccessibility of the protein digests. Proteins from chickpea sprouts showed a greater susceptibility to digestion with a 10% increase in alpha amino nitrogen. Peptides with potential immunoreactivity or bioactivity were catalogued in both digested chickpea sprouts and seeds using an in-silico approach. Peptides belonging to the non-specific transfer proteins, which are allergens in pulses, and peptides belonging to an IgE-binding hemagglutinin protein could only be identified in the digested chickpea sprouts. The observation collected paved the way to immune-based evaluations to assess the effect of germination on the allergenic potential.

Copper-64-Labeled Antibody Fragments for Immuno-PET/Radioimmunotherapy with Low Renal Radioactivity Levels and Amplified Tumor-Kidney Ratios.

Copper-64 (64Cu)-labeled antibody fragments such as Fab are useful for molecular imaging (immuno-PET) and radioimmunotherapy. However, these fragments cause high and persistent localization of radioactivity in the kidneys after injection. To solve this problem, this study assessed the applicability of a molecular design to 64Cu, which reduces renal radioactivity levels by liberating a urinary excretory radiometabolite from antibody fragments at the renal brush border membrane (BBM). Since 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) forms a stable complex with Cu, NOTA-conjugated Met-Val-Lys-maleimide (NOTA-MVK-Mal), which is a radio-gallium labeling agent for antibody fragments, was evaluated for applicability to 64Cu. The MVK linkage was recognized by the BBM enzymes to liberate [64Cu]Cu-NOTA-Met although the recognition of the MVK sequence for the [64Cu]Cu-NOTA-MVK derivative was reduced compared with that of its [67Ga]Ga-counterpart, probably due to the difference in the charge of the metal-NOTA complexes. When injected into mice, [64Cu]Cu-NOTA-MVK-Fab resulted in similar renal radioactivity levels to the 67Ga-labeled counterpart. In addition, [64Cu]Cu-NOTA-MVK-Fab resulted in lower renal radioactivity levels than those from 64Cu-labeled Fab using a conventional method, without a reduction in the tumor radioactivity levels. These findings indicate that our approach to reducing renal radioactivity levels by liberating a radiolabeled compound from antibody fragments at the renal BBM for urinary excretion is applicable to 64Cu-labeled antibody fragments and useful for immuno-PET and radioimmunotherapy.

Sub-fractions from Carica Papaya Seed Extracts Can Prevent Potassium Bromate- induced Changes in Activities of Renal Brush Border Membrane Enzymes and Some Enzymes of Carbohydrate Metabolism in the Kidney of Rats

Aim: To investigate the effect of the chromatographic fractions of Carica papaya seed on KBrO3 –induced reduction in the activities of renal brush border membrane (BBM) marker enzymes and the changes in activities of some enzymes of carbohydrate metabolism in the kidney of rats.&#x0D; Study Design: twenty male Wistar rats were divided into four groups, five rats per group; normal control, KBrO3 control, papaya fraction control and KBrO3 group administered with 126mg/kg body weight of the most active fraction of partially purified methanol extract of C. papaya for 48 hours.&#x0D; Place and Duration of Study: Department of Biochemistry Laboratory, Faculty of Basic Medical Sciences, Bayero University Kano, Nigeria.&#x0D; Methodology: The activities of renal BBM marker enzymes: γ-glutamyl transferase, alkaline phosphatase, maltase and leucine aminopeptidase were assayed in homogenates of renal cortex and medulla, and in brush border membrane vesicle (BBMV) isolated from cortex using standard methods. Furthermore, activities of the following enzymes representing different pathways of carbohydrate metabolism were determined in renal homogenates: hexokinase (HK), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), glucose 6-phosphatase (G6P), fructose 1,6-bisphosphatase (FBP), glucose 6-phosphate dehydrogenase (G6PD) and malic enzyme (ME).&#x0D; Results: KBrO3 administration significantly (P&lt;0.05) decreases the activities of all the BBM marker enzymes in renal homogenates and BBMV. It also decreases the activities of MDH, G6P, FBP and G6PD, and significantly increases (P&lt;0.05) that of HK, LDH and ME in renal homogenates however co-administration of most active fraction of C. papaya seed prevented all the KBrO3 -induced changes in these biochemical parameters.&#x0D; Conclusion: Chromatographic fractions of C. papaya seed extract possesses potent phytochemicals that could prevent KBrO3 –induced reduction in activities of renal BBM marker enzymes and the changes in enzymes of carbohydrate metabolism studied and therefore could be analyzed further to isolate the bioactive compounds.

Oral administration of thiram inhibits brush border membrane enzymes, oxidizes proteins and thiols, impairs redox system and causes histological changes in rat intestine: A dose dependent study

Pesticides are extensively employed worldwide, especially in agriculture to control weeds, insect infestation and diseases. Besides their targets, pesticides can also affect the health of non-target organisms, including humans The present study was conducted to study the effect of oral exposure of thiram, a dithiocarbamate fungicide, on the intestine of rats. Male rats were administered thiram at doses of 100, 250, 500 and 750 mg/kg body weight for 4 days. This treatment reduced cellular glutathione, total sulfhydryl groups but enhanced protein carbonyl content and hydrogen peroxide levels. In addition, the activities of all major antioxidant enzymes (catalase, thioredoxin reductase, glutathione peroxidase and glutathione-S-transferase) except superoxide dismutase were decreased. The antioxidant power of the intestine was impaired lowering the metal-reducing and free radical quenching ability. Administration of thiram also led to inhibition of intestinal brush border membrane enzymes, alkaline phosphatase, γ-glutamyl transferase, leucine aminopeptidase and sucrase. Activities of enzymes of pentose phosphate pathway, citric acid cycle, glycolysis and gluconeogenesis were also inhibited. Histopathology showed extensive damage in the intestine of thiram-treated rats at higher doses. All the observed effects were in a thiram dose-dependent manner. The results of this study show that thiram causes significant oxidative damage in the rat intestine which is associated with the marked impairment in the antioxidant defense system.