Intestinal Alkaline Phosphatase Research Articles

In Vitro and In Vivo Pharmacological Profiles of DS-1211, a Novel Potent, Selective, and Orally Bioavailable Tissue-Nonspecific Alkaline Phosphatase Inhibitor.

Inhibition of tissue-nonspecific alkaline phosphatase (TNAP) may prevent ectopic soft tissue calcification by increasing endogenous pyrophosphate (PPi). DS-1211 is a potent and selective novel small molecule TNAP inhibitor with well-characterized pharmacokinetics (PKs) in rodent and monkey. Herein, we report a comprehensive summary of studies establishing the pharmaceutical profile of DS-1211. In vitro studies characterized the mode of inhibition and inhibitory effects of DS-1211 on three human alkaline phosphatase (ALP) isozymes-TNAP, human intestinal ALP, human placental ALP-and on ALP activity across species in mouse, monkey, and human plasma. In vivo PK and pharmacodynamic (PD) effects of a single oral dose of DS-1211 in mice and monkeys were evaluated, including biomarker changes in PPi and pyridoxal 5'-phosphate (PLP). Oral bioavailability (BA) was determined through administration of DS-1211 at a 0.3-mg/kg dose in monkeys. In vitro experiments demonstrated DS-1211 inhibited ALP activity through an uncompetitive mode of action. DS-1211 exhibited TNAP selectivity and potent inhibition of TNAP across species. In vivo studies in mice and monkeys after single oral administration of DS-1211 showed linear PKs, with dose-dependent inhibition of ALP activity and increases in plasma PPi and PLP. Inhibitory effects of DS-1211 were consistent in both mouse and monkey. Mean absolute oral BA was 73.9%. Overall, in vitro and in vivo studies showed DS-1211 is a potent and selective TNAP inhibitor across species. Further in vivo pharmacology studies in ectopic calcification animal models and clinical investigations of DS-1211 in patient populations are warranted. © 2022 Daiichi Sankyo, Inc. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Zeta potential changing nanoemulsions based on a simple zwitterion

HypothesisSimple zwitterions used as auxiliary agents might have the potential to change the zeta potential of oil-in-water (o/w) nanoemulsions on the mucosa. ExperimentsThe zwitterion phosphorylated tyramine (p-Tyr) was synthesized by phosphorylation of Boc-tyramine (Boc-Tyr) using phosphoryl chloride (POCl3). It was incorporated with 2% (m/v) in a self-emulsifying lipophilic phase comprising Captex 35, Cremophor EL, Capmul MCM and glycerol 85 at a ratio of 30:30:30:10 v/v. Phosphate release and resulting change in zeta potential were evaluated by incubating p-Tyr containing nanoemulsion with isolated intestinal alkaline phosphatase (AP). Mucus permeating behavior was evaluated across mucus obtained from porcine small intestinal mucosa. Subsequently, cellular uptake studies were accomplished on Caco-2 cells. FindingsThe p-Tyr loaded nanoemulsion exhibited a mean droplet size of 43 ± 1.7 nm and zeta potential of −8.40 mV. Phosphate moieties were rapidly cleaved from p-Tyr loaded nanoemulsions after incubation with isolated AP resulting in a shift in zeta potential from −8.40 mV to +1.2 mV. p-Tyr loaded nanoemulsion revealed a significantly (p ≤ 0.001) improved mucus permeation compared to the same nanoemulsion having been pre-treated with AP. Cellular uptake of the zeta potential changing oily droplets was 2.4-fold improved. Phosphorylated zwitterions seem to be an alternative to cationic surfactants and considered as promising auxiliary agents for zeta potential changing nanoemulsions.

Intestinal alkaline phosphatase modulation by food components: predictive, preventive, and personalized strategies for novel treatment options in chronic kidney disease.

Alkaline phosphatase (AP) is a ubiquitous membrane-bound glycoprotein that catalyzes phosphate monoesters' hydrolysis from organic compounds, an essential process in cell signaling. Four AP isozymes have been described in humans, placental AP, germ cell AP, tissue nonspecific AP, and intestinal AP (IAP). IAP plays a crucial role in gut microbial homeostasis, nutrient uptake, and local and systemic inflammation, and its dysfunction is associated with persistent inflammatory disorders. AP is a strong predictor of mortality in the general population and patients with cardiovascular and chronic kidney disease (CKD). However, little is known about IAP modulation and its possible consequences in CKD, a disease characterized by gut microbiota imbalance and persistent low-grade inflammation. Mitigating inflammation and dysbiosis can prevent cardiovascular complications in patients with CKD, and monitoring factors such as IAP can be useful for predicting those complications. Here, we review IAP's role and the results of nutritional interventions targeting IAP in experimental models to prevent alterations in the gut microbiota, which could be a possible target of predictive, preventive, personalized medicine (PPPM) to avoid CKD complications. Microbiota and some nutrients may activate IAP, which seems to have a beneficial impact on health; however, data on CKD remains scarce.

Alterations in biochemical parameters of fish species under choline administration directly into the pond water in a semi-intensive fish farming system: A comparative study

The present study deals with the impact of choline chloride supplementation, applied directly into the pond water in a semi-intensive farm culture system for 90 d on two Indian Major Carps (IMCs) (Catla catla, Labeo rohita) and two air-breathing teleostean fishes (Anabas testudineus, Clarias batracus).The experiment was conducted in two seasons (breeding and dry) under choline (treated) and non-choline (control) exposure. Biochemical parameters, viz., PRO (total protein) content gained maximum significantly (p< 0.01) in treated-breeding with the lowest in control-dry in intestine, liver, muscle, brain. IMCs showed significant (p< 0.01) increased amylase activity in the liver and intestine in treated-dry and treatment-breeding conditions, while liver amylase activity in both the seasons increased manifolds significantly (p< 0.01) in air-breathing fishes, showing a sharp decline only in the intestine. Protease and lipase activity in the intestine and liver of choline-fed fishes disclosed declining trend in the breeding season significantly (p< 0.01), but treated (choline-fed) IMCs under dry revealed the significant (p< 0.01) increasing and decreasing trend of protease and lipase activity respectively; the reverse trend at dry season was found in air-breathing fishes under choline-fed condition. ALP (alkaline phosphatase) in intestine indicated significant (p< 0.01) lowest activity in treatment-breeding, highest in treatment-dry. This study explored the higher activity towards total protein generation, especially muscle; higher carbohydrate digestion; elevated lipid digestion in choline-fed fishes. So, the fishes from this farm-based pisciculture system are healthy and its yield can be prescribed as a rich source of quality fish food for human health.

Gastrointestinal manifestations after Roux-en-Y gastric bypass surgery in individuals with and without type 2 diabetes.

Roux-en-Y gastric bypass (RYGB) surgery is an effective treatment for obesity, which improves cardiovascular health and reduces the risk of premature mortality. However, some reports have suggested that RYGB may predispose patients to adverse health outcomes, such as inflammatory bowel disease (IBD) and colorectal cancer. The present prospective study aimed to evaluate the impact of RYGB surgery on cardiovascular risk factors and gastrointestinal inflammation in individuals with and without type 2 diabetes (T2D). University hospital setting in Finland. Blood and fecal samples were collected at baseline and 6 months after surgery from 30 individuals, of which 16 had T2D and 14 were nondiabetics. There were also single study visits for 6 healthy reference patients. Changes in cardiovascular risk factors, serum cholesterol, and triglycerides were investigated before and after surgery. Fecal samples were analyzed for calprotectin, anti-Saccharomyces cerevisiae immunoglobulin A antibodies (ASCA), active lipopolysaccharide (LPS) concentration, short-chain fatty acids (SCFAs), intestinal alkaline phosphatase activity, and methylglyoxal-hydro-imidazolone (MG-H1) protein adducts formation. After RYGB, weight decreased on average -21.6% (-27.2 ± 7.8 kg), excess weight loss averaged 51%, and there were improvements in cardiovascular risk factors. Fecal calprotectin levels (P < .001), active LPS concentration (P < .002), ASCA (P < .02), and MG-H1 (P < .02) values increased significantly, whereas fecal SCFAs, especially acetate (P < .002) and butyrate (P < .03) levels, were significantly lowered. The intestinal homeostasis is altered after RYGB, with several fecal markers suggesting increased inflammation; however, clinical significance of the detected changes is currently uncertain. As chronic inflammation may predispose patients to adverse health effects, our findings may have relevance for the suggested association between RYGB and increased risks of incident IBD and colorectal cancer.

The effects of dietary extract of mulberry leaf on growth performance, hypoxia-reoxygenation stress and biochemical parameters in various organs of fish

The study explored the effects of extract of mulberry leaves (EML) on the growth performance and hypoxia-reoxygenation stress as well as biochemical parameters in various organs of crucian carp (Carassius auratus gibelio). The results indicated that dietary EML improved the growth of crucian carp. The appropriate concentration of EML for fish growth was estimated to be 46.93 g kg−1 diet. Moreover, dietary EML raised the digestive, absorptive and metabolic function by improving feed intake and alpha‐amylase, lipase, trypsin, alkaline phosphatase, glutamate-oxaloacetate transaminase (GOT) and glutamate-pyruvate transaminase activities in hepatopancreas or intestine and decreasing plasma ammonia content, and respiratory function by increasing GOT activities in the branchiae and hemoglobin level in red blood cells of crucian carp. After 30 days feeding trial, crucian carp was treated with hypoxia-reoxygenation. The result showed that dietary EML improved hypoxia tolerance in crucian carp. The appropriate concentration of EML for hypoxia tolerance was estimated to be 57.21 g kg−1 diet. Hypoxia-reoxygenation stimulated the loss of partial digestive, absorptive, respiratory and metabolic function, reactive oxygen species (ROS) generation and cellular component oxidation in the digestive and respiratory organs, suggesting the ability of hypoxia-reoxygenation to induce oxidative lesion in fish. However, dietary EML reduced the oxidative lesion in a way of suppressing the production of ROS and oxidation of cellular component and elevating activities of enzymatic antioxidant and levels of non-enzymatic antioxidant in the digestive and respiratory organs of fish caused by hypoxia-reoxygenation. Therefore, EML can be utilized as an inhibitor of hypoxia-reoxygenation stress in fish.

Targeting the gut to prevent sepsis from a cutaneous burn.

Severe burn injury induces gut barrier dysfunction and subsequently a profound systemic inflammatory response. In the present study, we examined the role of the small intestinal brush border enzyme, intestinal alkaline phosphatase (IAP), in preserving gut barrier function and preventing systemic inflammation after burn wound infection in mice. Mice were subjected to a 30% total body surface area dorsal burn with or without intradermal injection of Pseudomonas aeruginosa. Mice were gavaged with 2000 units of IAP or vehicle at 3 and 12 hours after the insult. We found that both endogenously produced and exogenously supplemented IAP significantly reduced gut barrier damage, decreased bacterial translocation to the systemic organs, attenuated systemic inflammation, and improved survival in this burn wound infection model. IAP attenuated liver inflammation and reduced the proinflammatory characteristics of portal serum. Furthermore, we found that intestinal luminal contents of burn wound–infected mice negatively impacted the intestinal epithelial integrity compared with luminal contents of control mice and that IAP supplementation preserved monolayer integrity. These results indicate that oral IAP therapy may represent an approach to preserving gut barrier function, blocking proinflammatory triggers from entering the portal system, preventing gut-induced systemic inflammation, and improving survival after severe burn injuries.

The Effect of Overexpression of Intestinal Alkaline Phosphatase (IAP) on Intestinal Permeability and Renal Failure in Lipopolysaccharide (LPS) Treated IAP Transgenic (IAPTg) Mice

The Effect of Overexpression of Intestinal Alkaline Phosphatase (IAP) on Intestinal Permeability and Renal Failure in Lipopolysaccharide (LPS) Treated IAP Transgenic (IAPTg) Mice

S1P Lyase siRNA Dampens Malignancy of DLD-1 Colorectal Cancer Cells.

Sphingosine-1-phosphate lyase 1 (S1P lyase or SGPL1) is an essential sphingosine-1-phosphate-degrading enzyme. Its manipulation favors onset and progression of colorectal cancer and others in vivo. Thus, SGPL1 is an important modulator of cancer initiation. However, in established cancer, the impact of retrospective SGPL1 modulation is elusive. Herein, we analyzed how SGPL1 siRNA affects malignancy of the human colorectal cancer cells DLD-1 and found that in parallel to the reduction of SGPL1 expression levels, migration, invasion, and differentiation status changed. Diminished SGPL1 expression was accompanied with reduced cell migration and cell invasion in scratch assays and transwell assays, whereas metabolic activity and proliferation was not altered. Decreased migration was attended by increased cell-cell-adhesion through upregulation of E-cadherin and formation of cadherin-actin complexes. Spreading cell islets showed lower vimentin abundance in border cells. Furthermore, SGPL1 siRNA treatment induced expression of epithelial cell differentiation markers, such as intestinal alkaline phosphatase and cytokeratin 20. Hence, interference with SGPL1 expression augmented a partial redifferentiation of colorectal cancer cells toward normal colon epithelial cells. Our investigation showed that SGPL1 siRNA influenced tumorigenic activity of established colorectal cancer cells. We therefore suggest SGPL1 as a target for lowering malignant potential of already existing cancer.

IIAEK Targets Intestinal Alkaline Phosphatase (IAP) to Improve Cholesterol Metabolism with a Specific Activation of IAP and Downregulation of ABCA1.

IIAEK (Ile-Ile-Ala-Glu-Lys, lactostatin) is a novel cholesterol-lowering pentapeptide derived from bovine milk β-lactoglobulin. However, the molecular mechanisms underlying the IIAEK-mediated suppression of intestinal cholesterol absorption are unknown. Therefore, we evaluated the effects of IIAEK on intestinal cholesterol metabolism in a human intestinal model using Caco-2 cells. We found that IIAEK significantly reduced the expression of intestinal cholesterol metabolism-associated genes, particularly that of the ATP-binding cassette transporter A1 (ABCA1). Subsequently, we chemically synthesized a novel molecular probe, IIXEK, which can visualize a complex of target proteins interacting with photoaffinity-labeled IIAEK by fluorescent substances. Through photoaffinity labeling and MS analysis with IIXEK for the rat small intestinal mucosa and intestinal lipid raft fractions of Caco-2 cells, we identified intestinal alkaline phosphatase (IAP) as a specific molecule interacting with IIAEK and discovered the common IIAEK-binding amino acid sequence, GFYLFVEGGR. IIAEK significantly increased IAP mRNA and protein levels while decreasing ABCA1 mRNA and protein levels in Caco-2 cells. In conclusion, we found that IIAEK targets IAP to improve cholesterol metabolism via a novel signaling pathway involving the specific activation of IAP and downregulation of intestinal ABCA1.

Peripartum changes in serum activities of three major alkaline phosphatase isoenzymes in Holstein dairy cows.

This study investigated changes in serum levels of hepatic, bone, and intestinal alkaline phosphatase (ALP) isoenzymes (ALP2, ALP3, and ALP5, respectively) in Holstein cows around parturition. Tartrate-resistant acid phosphatase 5b (TRAP5b) activity and calcium (Ca) concen-trations were also measured. We analyzed blood samples from 11 late-pregnant heifers (primipa-rous group) and 13 multiparous (2-4 lactations; multiparous group) cows at 3 weeks (18-24 days prepartum; -3 weeks), 2 weeks (17-11 days prepartum; -2 weeks), and 1 week (10-4 days prepar-tum; -1 weeks) before parturition; the day of calving (within 12 h post-calving; day 0); and 5 days postpartum (5 days). ALP3 activity was significantly higher in the primiparous group than in the multiparous group, whereas the activities decreased significantly in both groups after 5 days. ALP2 and ALP5 activities did not change, whereas ALP2 activity was significantly higher in the primiparous group than in the multiparous group. TRAP5b activity was significantly higher in the primiparous group than in the multiparous group and showed a transient significant increase at day 0. Ca concentration significantly decreased at day 0 in both groups; the Ca level at day 0 was significantly higher in the primiparous group than in the multiparous group. These data show that ALP3 activity in serum may indicate a change in osteoblastic bone forma-tion after calving, but further study is needed to determine the clinical application for measuring ALP isoenzymes in bovine medicine.

Physical processing or supplementation of feeds with phytogenic compounds, alginate oligosaccharide or nucleotides as methods to improve the utilization of Gracilaria gracilis by juvenile European seabass (Dicentrarchus labrax)

This study assessed both the effectiveness of a physical-mechanical rupture method and the ability of feed additives (phytogenic compounds, alginate oligosaccharide and nucleotides) to enhance the utilization of G. gracilis by European seabass. A commercial-based diet was used as control diet (CTRL) and compared with five isoproteic (53.5% Dry matter, DM) and isolipidic (14.9% DM) diets containing 8% of G. gracilis. This seaweed was either unprocessed (diet GRA) or subjected to physical processing (diet GRAP). The three additive-containing diets were formulated by supplementing the GRA diet with either 0.02% phytogenic compounds (PHY), 2.5% oligo-alginate (OAS) or 0.08% free nucleotides (NUC). Triplicate groups of nineteen fish (29.7 ± 0.02 g) were distributed by 50 L tanks (11.3 kg m−3) and fed the experimental diets to satiety during 106 days. By the end of the trial, growth performance and nutrient utilization (specific growth ratio, feed conversion ratio, apparent digestibility coefficients, nutrient balance, intestinal brush border membrane enzyme activities and plasma metabolic parameters), gut histomorphology, antioxidant and immunological status of fish were evaluated. The ability of fish to digest seaweed-rich diets was largely improved by the technological processing of G. gracilis, albeit nil effect on fish specific growth rate (1.0 in all groups). This major achievement was associated with increased ability of GRAP to digest protein (84 vs 68% in GRA) and energy (64 vs 38% in GRA). The use of feed additives in Gracilaria-rich diets was less efficacious in improving European sea bass nutrient and energy ADCs, but have still improved the overall digestibility of those diets. Fish fed alginate oligosaccharide was mainly associated with increased activity of anterior intestine enzymes, particularly intestinal alkaline phosphatase (IAP; 174.4 vs 104.7–120.6 μm min−1 g−1 in Gracilaria-rich diets). Moreover, the algae technological processing and both the nucleotides and the alginate oligosaccharide seem to have positively affected the intestinal villus width compared to the negative impact seen in fish fed GRA. The tested additives had limited impact on oxidative stress, although glutathione peroxidase (GPx; 2.1 μmol min−1 mg protein−1) and catalase (CAT; 35 μmol min−1 mg protein−1) activities were lowest in fish fed NUC and PHY, respectively. It can be concluded that the physical processing of Gracilaria sp. or the addition of either oligo-alginate or nucleotides can effectively increase the nutritional value of this seaweed for European seabass diets.

Prodrug Strategies to Improve the Solubility of the HCV NS5A Inhibitor Pibrentasvir (ABT-530).

A research program to discover solubilizing prodrugs of the HCV NS5A inhibitor pibrentasvir (PIB) identified phosphomethyl analog 2 and trimethyl-lock (TML) prodrug 9. The prodrug moiety is attached to a benzimidazole nitrogen atom via an oxymethyl linkage to allow for rapid and complete release of the drug for absorption following phosphate removal by intestinal alkaline phosphatase. These prodrugs have good hydrolytic stability properties and improved solubility compared to PIB, both in aqueous buffer (pH 7) and FESSIF (pH 5). TML prodrug 9 provided superior in vivo performance, delivering high plasma concentrations of PIB in PK studies conducted in mice, dogs, and monkeys. The improved dissolution properties of these phosphate prodrugs provide them the potential to simplify drug dosage forms for PIB-containing HCV therapy.

A summary of feed additives, intestinal health and intestinal alkaline phosphatase in piglet nutrition

Weaning is considered the “critical window” in the piglet’s life because it is associated with several stress factors, such as loss of contact with the mother and original litter, solid diet, environmental and structural changes, and the establishment of a new hierarchy. During this abrupt period, several events such as reduced feed intake, high morbidity, susceptibility to enteric infections and post-weaning diarrhoea are observed. The nutritional landscape of the piglet gut is modified, which can compromise the maturity of the gastrointestinal system, the stable intestinal microbiome and the active immunity developed as an indicator of intestinal health. However, with increased awareness of feed safety issues and the development of drug-resistant bacteria, the interest in producing pigs without the use of antimicrobial growth promoters (AGP) is increasing, since long-term use and therapeutic doses of AGP can contribute to the reduction of bacterial diversity and increase of inflammatory bowel disease (IBD). Thus, the most widely researched alternatives include the use of feed additives, feeding strategies, nutraceuticals/functional foods and available handling that can reduce the risk of IBD beyond basic nutritional functions. Studies have reported intestinal alkaline phosphatase as a new nutritional therapy associated with intestinal health which may be a “key additive” in the AGP replacement. In this review article, the purpose is to show some current aspects of feed additive research, addressing a concept of the “intestinal health” from different points of view and properties of alkaline phosphatase.

Understanding the enzymatic inhibition of intestinal alkaline phosphatase by aminophenazone-derived aryl thioureas with aided computational molecular dynamics simulations: synthesis, characterization, SAR and kinetic profiling.

The work presented in this paper aims toward the synthesis of aryl thiourea derivatives 4a-l of pyrazole based nonsteroidal anti-inflammatory drug named 4-aminophenazone, as potential inhibitors of intestinal alkaline phosphatase enzyme. The screening of synthesized target compounds 4a-l for unraveling the anti-inflammatory potential against calf intestinal alkaline phosphatase gives rise to lead member 4c possessing IC50 value 0.420 ± 0.012µM, many folds better than reference standard used (KH2PO4 IC50 = 2.8 ± 0.06µM and L-phenylalanine IC50 = 100 ± 3.1µM). SAR for unfolding the active site binding pocket interaction along with the mode of enzyme inhibition based on kinetic studies is carried out which showed non-competitive binding mode. The enzyme inhibition studies were further supplemented by molecular dynamic simulations for predicting the protein behavior against active inhibitors 4c and 4g during docking analysis. The preliminary toxicity of the synthesized compounds was determined by using brine shrimp assay. This work also includes detailed biochemical analysis along with RO5 parameters for all the newly synthesized drug derivatives 4a-l.

Excreta biomarkers in response to different gut barrier dysfunction models and probiotic supplementation in broiler chickens.

Increased intestinal permeability (IP) and inflammation are both linked with functionality of the intestinal barrier and in particular enterocytes. Currently, almost all assessment methods of the intestinal barrier function are invasive. The present study aimed to quantify selected proteins as novel biomarkers in excreta of broiler chickens to facilitate non-invasive assessment of gut barrier function using enzyme-linked immunosorbent assays (ELISA). It was further hypothesised that probiotics as feed additives may counteract gut barrier dysfunction. A 3 × 2 factorial arrangement of treatments was used with the main factors being gut barrier dysfunction models (control, rye-based diet, and dexamethasone–DEX) with and without probiotic supplementation (a three-strain Bacillus) using 72 male Ross 308 day-old chickens. Each of the 6 experimental treatments was replicated 12 times. On d 21 of age, fluorescein isothiocyanate dextran (FITC-d) uptake into serum was examined to test IP. Fresh excreta samples were collected on d 20. The biomarkers included alpha-1 antitrypsin (A1AT), intestinal fatty acid binding protein (I-FABP), lipocalin-2 (LCN2), fibronectin (FN), intestinal alkaline phosphatase (IAP), ovotransferrin (OVT) and superoxide dismutase [Cu-Zn] (SOD1). Only DEX increased (P<0.001) FITC-d passage to the blood on d 21 of age, indicating a greater IP. The excreta concentrations of A1AT, I-FABP and SOD1 were unaltered by the experimental treatments. DEX increased (P<0.05) FN concentration in excreta compared with control birds. Conversely, inclusion of rye in the diet reduced (P<0.05) FN but increased (P<0.001) OVT in excreta. Independently, DEX decreased IAP (P<0.05) in excreta compared with control and rye-fed birds. The excreta concentration of LCN2 tended (P = 0.086) to increase in birds injected by DEX. There was no demonstrable effect of probiotic addition on any of the studied parameters. Among the tested biomarkers, FN, IAP, and LCN2 revealed promise as biomarkers of intestinal barrier function quantified by ELISA kits.

Bacteria Remediate the Effects of Food Additives on Intestinal Function in an in vitro Model of the Gastrointestinal Tract.

As the site of nutrient absorption, the small intestine is continuously exposed to preservatives and additives present in consumed food. While the effects of diet on the lower gastrointestinal tract are widely studied, the effects of food additives on the small intestinal epithelium and microbiota are less clearly understood. The goal of this work was to develop and establish a physiologically relevant model of the upper gastrointestinal tract to study the complex interactions between food additives, individual bacterial species, and intestinal function. To achieve this, an in vitro model incorporating simulated digestion, human intestinal epithelial cells, and the commensal, Gram-positive Lactobacillus rhamnosus, or the opportunistic, Gram-negative Escherichia coli was developed. This model was used to assess intestinal permeability and alkaline phosphatase activity following exposure to high glucose (HG), salt, emulsifier (TWEEN 20), food (milk chocolate candies) or chemical grade titanium dioxide nanoparticles (TiO2-NP), and food (whole wheat bread) or chemical grade gluten. It was found that HG increased intestinal permeability, the presence of bacteria remediated the negative effects of HG on intestinal permeability, and a decrease in permeability and IAP activity was observed with increasing concentration of TWEEN 20 both in the presence and absence of bacteria. While L. rhamnosus influenced the activity of intestinal alkaline phosphatase and tight junction protein distribution, E. coli produced indole to reinstate intestinal permeability. The source of TiO2 and gluten led to altered impacts on permeability and IAP activity. The growth of E. coli and L. rhamnosus was found to depend on the type of food additive used. Overall, the presence of bacteria in the in vitro model influenced the effects of food additives on intestinal function, suggesting a complex association between diet and upper GI microbiota. This model provides a method to study small intestinal function and host-microbe interactions in vitro in both healthy and diseased conditions.

Tissue alkaline phosphatase activity and expression in an experimental infant swine model of cardiopulmonary bypass with deep hypothermic circulatory arrest

BackgroundInfant cardiac surgery with cardiopulmonary bypass results in decreased circulating alkaline phosphatase that is associated with poor postoperative outcomes. Bovine intestinal alkaline phosphatase infusion represents a novel therapy for post-cardiac surgery organ injury. However, the effects of cardiopulmonary bypass and bovine-intestinal alkaline phosphatase infusion on tissue-level alkaline phosphatase activity/expression are unknown.MethodsInfant pigs (n = 20) underwent cardiopulmonary bypass with deep hypothermic circulatory arrest followed by four hours of intensive care. Seven control animals underwent mechanical ventilation only. Cardiopulmonary bypass/deep hypothermic circulatory arrest animals were given escalating doses of bovine intestinal alkaline phosphatase infusion (0-25 U/kg/hr.; n = 5/dose). Kidney, liver, ileum, jejunum, colon, heart and lung were collected for measurement of tissue alkaline phosphatase activity and mRNA.ResultsTissue alkaline phosphatase activity varied significantly across organs with the highest levels found in the kidney and small intestine. Cardiopulmonary bypass with deep hypothermic circulatory arrest resulted in decreased kidney alkaline phosphatase activity and increased lung alkaline phosphatase activity, with no significant changes in the other organs. Alkaline phosphatase mRNA expression was increased in both the lung and the ileum. The highest dose of bovine intestinal alkaline phosphatase resulted in increased kidney and liver tissue alkaline phosphatase activity.ConclusionsChanges in alkaline phosphatase activity after cardiopulmonary bypass with deep hypothermic circulatory arrest and bovine intestinal alkaline phosphatase delivery are tissue specific. Kidneys, lung, and ileal alkaline phosphatase appear most affected by cardiopulmonary bypass with deep hypothermic circulatory arrest and further research is warranted to determine the mechanism and biologic importance of these changes.

Synthesis, characterization, alkaline phosphatase inhibition assay and molecular modeling studies of 1-benzylidene-2-(4-tert- butylthiazol-2-yl) hydrazines

Alkaline phosphatases are homodimeric protein enzymes which removes phosphates from several types of molecules. These catalyze the hydrolysis of monoesters in phosphoric acid which in turn catalyze a transphosphorylation reaction. Thiazoles are a privileged class of heterocyclic compounds which may potentially serve as effective phosphatase inhibitors. In this regard, the present research paper reports the facile synthesis and characterization of substituted 1-benzylidene-2-(4-tert-butylthiazol-2-yl) hydrazines with excellent yields. The synthesized compounds were tested for inhibitory potential against alkaline phosphatases. The compound 1-(4-Hydroxy, 3-methoxybenzylidene)-2-(4-tert-butylthiazol-2-yl) hydrazine (5e) was found to be the most potent inhibitor of human tissue non-alkaline phosphatase in this group of molecules with an IC50 value of 1.09 ± 0.18 µM. The compound 1-(3,4-dimethoxybenzylidene)-2-(4-tert-butylthiazol-2-yl) hydrazine (5d) exhibited selectivity and potency for human intestinal alkaline phosphatase with an IC50 value of 0.71 ± 0.02 µM. In addition, structure activity relationship and molecular docking studies were performed to evaluate their binding modes with the target site of alkaline phosphatase. The docking analysis revealed that the most active inhibitors showed the important interactions within the binding pockets of human intestinal alkaline phosphatase and human tissue non-alkaline phosphatase and may be responsible for the inhibitory activity of the compound towards the enzymes. Therefore, the screened thiazole derivatives provided an outstanding platform for further development of alkaline phosphatase inhibitors.

Two farnesyl pyrophosphate synthases, GhFPS1–2, in Gossypium hirsutum are involved in the biosynthesis of farnesol to attract parasitoid wasps

Sesquiterpenoids play an import role in the direct or indirect defense of plants. Farnesyl pyrophosphate synthases (FPSs) catalyze the biosynthesis of farnesyl pyrophosphate, which is a key precursor of farnesol and (E)-β-farnesene. In the current study, two FPS genes in Gossypium hirsutum, GhFPS1 and GhFPS2, were heterologously cloned and functionally characterized in a greenhouse setting. The open reading frames for full-length GhFPS1 and GhFPS2 were each 1 029 nucleotides, and encoded two proteins of 342 amino acids with molecular weights of 39.4 kDa. The deduced amino acid sequences of GhFPS1–2 showed high identity to FPSs of other plants. Quantitative real-time PCR analysis revealed that GhFPS1 and GhFPS2 were highly expressed in G. hirsutum leaves, and were upregulated in methyl jasmonate (MeJA)-, methyl salicylate (MeSA)- and aphid infestation-treated cotton plants. The recombinant proteins of either GhFPS1 or GhFPS2 plus calf intestinal alkaline phosphatase could convert geranyl diphosphate (GPP) or isopentenyl diphosphate (IPP) to one major product, farnesol. Moreover, in electrophysiological response and Y-tube olfactometer assays, farnesol showed obvious attractiveness to female Aphidius gifuensis, which is an important parasitic wasp of aphids. Our findings suggest that two GhFPSs are involved in farnesol biosynthesis and they play a crucial role in indirect defense of cotton against aphid infestation.