Conjugated Bile Salts Research Articles

Bile salt tolerance of Lactococcus lactis is enhanced by expression of bile salt hydrolase thereby producing less bile acid in the cells.

Changes of bile salt tolerance, morphology and amount of bile acid within cells were studied to evaluate the exact effects of bile salt hydrolase (BSH) on bile salt tolerance of microorganism. The effect of BSHs on the bile salt tolerance of Lactococcus lactis was examined by expressing two BSHs (BSH1 and BSH2). Growth of L. lactis expressing BSH1 or BSH2 was better under bile salt stress compared to wild-type L. lactis. As indicated by transmission electron microscopy, bile acids released by the action of BSH induced the formation of micelles around the membrane surface of cells subject to conjugated bile salt stress. A similar micelle containing bile acid was observed in the cytoplasm by liquid chromatography-mass spectrometry. BSH1 produced fewer bile acid micelles in the cytoplasm and achieved better cell growth of L. lactis compared to BSH2. Expression of BSH improved bile salt tolerance of L. lactis but excessive production by BSH of bile acid micelles in the cytoplasm inhibited cell growth.

Effect of taurine on intestinal recovery following intestinal ischemia-reperfusion injury in a rat.

Taurine (TAU) is a sulfur-containing amino acid that is involved in a diverse array of biological and physiological functions, including bile salt conjugation, osmoregulation, membrane stabilization, calcium modulation, anti-oxidation, and immunomodulation. Several studies have established that treatment with TAU significantly protects cerebral, cardiac and testicular injury from ischemia-reperfusion (IR). The purpose of the present study was to examine the effect of TAU on intestinal recovery and enterocyte turnover after intestinal IR injury in rats. Male Sprague-Dawley rats were divided into four experimental groups: (1) Sham rats that underwent laparotomy, (2) Sham-TAU rats that underwent laparotomy and were treated with intraperitoneal (IP) TAU (250mg/kg); (3) IR-rats that underwent occlusion of both superior mesenteric artery and portal vein for 30min followed by 48h of reperfusion, and (4) IR-TAU rats that underwent IR and were treated with IP TAU (250mg/kg) immediately before abdominal closure. Intestinal structural changes, Park's injury score, enterocyte proliferation and enterocyte apoptosis were determined 24h following IR. The expression of Bax, Bcl-2, p-ERK and caspase-3 in the intestinal mucosa was determined using Western blot and immunohistochemistry. Treatment with TAU resulted in a significant decrease in Park's injury score compared to IR animals. IR-TAU rats also demonstrated a significant increase in mucosal weight in jejunum and ileum, villus height in jejunum and ileum and crypt depth in ileum compared to IR animals. IR-TAU rats also experienced significantly lower apoptotic indices in jejunum and ileum which was accompanied by a higher Bcl-2/Bax ratio compared to IR animals. Treatment with taurine prevents gut mucosal damage and inhibits intestinal epithelial cell apoptosis following intestinal IR in a rat.

Role of taurine in the pathogenesis of obesity.

Taurine is a sulfur-containing amino acid that is present in mammalian tissues in millimolar concentrations. Taurine is involved in a diverse array of biological and physiological functions, including bile salt conjugation, osmoregulation, membrane stabilization, calcium modulation, anti-oxidation, and immunomodulation. The prevalence of obesity and being overweight continues to rise worldwide at an alarming rate. Obesity is associated with a higher risk of metabolic and cardiovascular diseases, cancer, and other clinical conditions. Ingestion of taurine has been shown to alleviate metabolic diseases such as hyperlipidemia, diabetes, hypertension, and obesity in animal models. A global epidemiological survey showed that 24-h urinary taurine excretion, as a marker of dietary taurine intake, was inversely associated with BMI, blood pressure, and plasma cholesterol in humans. In addition, taurine chloramine, an endogenous product derived from activated neutrophils, has been reported to suppress obesity-induced oxidative stress and inflammation in adipocytes. Synthetic activity and concentration of taurine in adipose tissues and plasma have been shown to decrease in humans and animals during the development of obesity, suggesting a relationship between taurine deficiency and obesity. In this review, I summarize the effects of taurine on the progression of obesity in animal models and humans. Furthermore, I discuss possible mechanisms underlying the antiobesity effects of taurine.

Ontogenetic taurine biosynthesis ability in rainbow trout (Oncorhynchus mykiss).

Taurine (2-aminoethane sulfonic acid) plays important roles in multiple physiological processes including osmoregulation, bile salt conjugation and membrane protection. It is known that taurine biosynthesis varies in different fish species. However, its ontogenetic regulation has not been clear. In the present study, we found that the hepatic concentrations of taurine increased marginally with rainbow trout growth. The mRNA expression, protein levels and enzyme activities of key enzymes involved in taurine biosynthesis, cysteine dioxygenase (CDO) and cysteine sulfinate decarboxylase (CSD), were analyzed. Our results showed that the mRNA levels and protein abundances of CSD increased dramatically with the development of rainbow trout stages while the enzyme activities showed a slight improvement. However, the expression and activities of CDO decreased with rainbow trout growth. These results provide valuable information on defining the exact supplementation of taurine in diets for different stages of rainbow trout and give new insights into elucidating the regulation of taurine metabolism in rainbow trout.

Hepatology highlights

Hepatology highlights

Taurine: a critical nutrient for future fish feeds

Taurine is a sulfonic acid found in high concentrations in animal tissues. In recent years, a number of studies have demonstrated the essentiality of dietary taurine for many commercially relevant species, especially marine teleosts. Consequently, the removal of taurine-rich dietary ingredients such as fishmeal may create a deficiency, of which symptoms include reduced growth and survival, increased susceptibility to diseases, and impaired larval development. These symptoms emphasize the systemic role of taurine in the animal’s physiology and provide few clues as to the underlying mechanisms of taurine function. In fact, a myriad of roles have been attributed to taurine in mammals, ranging from bile salt conjugation to membrane stabilization, osmoregulation, anti-oxidation, immunomodulation, calcium-signaling, and neuroprotection. This review describes the current knowledge of taurine physiology and metabolism in fish and requirement levels in relevant species, and highlights possible parallels with mammalian taurine metabolism. In addition, the effects of ingredient processing and feed manufacturing on taurine bioavailability are discussed. Finally, regulatory aspects are brought to the forefront: although the supplementation of taurine will be necessary to further reduce the use of ingredients such as fishmeal, taurine is not currently approved by the FDA in the USA for fish feeds. Obtaining approval in the United States to utilize taurine in fish feeds can improve the environmental and economic sustainability of fish feeds nation-wide. • In susceptible species, dietary taurine must be supplemented when using taurine-poor ingredients in feeds. • However, taurine is currently not listed as an approved ingredient for animal feeds in the USA. • Taurine is involved in bile salt formation, membrane stability, immunomodulation, anti-oxidation and mitochondrial function, and calcium-signaling. • The roles of taurine and its mechanisms in fish remain poorly described.

Probing Interactions between β-Glucan and Bile Salts at Atomic Detail by 1H–13C NMR Assays

Polysaccharides are prospective hosts for the delivery and sequestration of bioactive guest molecules. Polysaccharides of dietary fiber, specifically cereal (1 → 3)(1 → 4)-β-glucans, play a role in lowering the blood plasma cholesterol level in humans. Direct host-guest interactions between β-glucans and conjugated bile salts are among the possible molecular mechanisms explaining the hypocholesterolemic effects of β-glucans. The present study shows that (1)H-(13)C NMR assays on a time scale of minutes detect minute signal changes in both bile salts and β-glucans, thus indicating dynamic interactions between bile salts and β-glucans. Experiments are consistent with stronger interactions at pH 5.3 than at pH 6.5 in this in vitro assay. The changes in bile salt and β-glucan signals suggest a stabilization of bile salt micelles and concomitant conformational changes in β-glucans.

Role of primary bile salts in the regulation of sinusoidal substrate uptake in rat liver

Background Bile salts undergo enterohepatic circulation which is essential for bile salt homeostasis. From the biliary tract, they are excreted into the small intestine, absorbed into the blood and transported back to the liver. The sinusoidal uptake systems of the liver efficiently extract bile salts and other substrates from portal blood. In rat liver, these systems include Ntcp (sodium taurocholate cotransporting polypeptide) and the organic anion transporting polypeptides Oatp1a1, Oatp1a4 and Oatp1b2. While Ntcp represents the major basolateral uptake transporter for conjugated bile salts, the Oatps have broad substrate spectra including estrogenor leukotriene-conjugates. Their transport function is regulated by long-term and short-term mechanisms. Long-term adaptation of bile salt transporters involve changes at the level of gene expression and transporter degradation, while short-term regulation includes covalent transporter modifications, substrate availability and rapid endoand exocytosis of transporter-containing vesicles (reviewed in [1]). Ntcp as well as Oatp1a1 and Oatp1a4 underlie short-term control which has been demonstrated in several in vitro models. Hypoosmolarity or cAMP for instance increase the Ntcp-dependent bile salt uptake (increase of Vmax) within minutes by translocation of intracellular Ntcp to the plasma membrane [2,3]. In contrast, rapid clathrin-dependent endocytosis of Ntcp was demonstrated only recently by activation of PKC with phorbolesters [4,5]. However, to date in vivo-studies on short-term regulation of basolateral transporters are rare. Bile salt inflow to the liver may increase considerably under pathophysiological conditions or postprandial under physiological conditions [6]. To date, the interrelation between substrate load and transport activity is largely unknown. Therefore, the aim of this study was to investigate the role of primary bile salts in short-term feedback regulation of sinusoidal substrate uptake in perfused rat liver.

Su1252 Gastric Juice Composition and Acid Suppression in Pediatric Gastroesophageal Reflux Disease

INTRODUCTION: Gastric acid suppression is justified to prevent severe gastro esophageal reflux (GER) disease related complications. However, it does not reduce the total amount and proximal extent of GER in the esophagus and non-acid components are able to induce (extra-esophageal) GER symptoms as well. No data on the composition of gastric juice (GJ) in children using gastric acid suppression exists. We therefore aimed to assess whether the composition of gastric juice in children using proton pump inhibitors (PPIs) differs, compared to that of their controls. METHODS: Infants and children (0-18 years) on proton pump inhibitors (PPIs) for at least six weeks and a control group not using anti reflux medication, were included. GJ was obtained through an existing nasogastric or a percutaneous endoscopic gastrostomy tube/ Mic-key gastrostomy. In the collected GJ (5 ml), pH, pepsin activity, bile salts and endotoxin (LPS) levels were determined. Pepsin was measured using a fluorometric assay using 4-Methyl-Coumaryl-7-Amide (MCA) substrate with/without pepstatin. Concentrations of deconjugated and taurine/glycine-conjugated bile salts were assessed by reversephase HPLC. Levels of LPS were determined using the spectrophotometric Limulus Amebocyte Lysate assay. RESULTS: GJ was analyzed from 16 children with (median: 3.8 yrs, range: 17.6 years) and 16 children (4.0 yrs, range: 16.0) without PPI therapy. Median duration of PPI treatment was 24 weeks (range: 514 weeks). Gastric pH was 5.0 (range: 5.0) and 1.0 (range: 4.5) in the PPI and control group respectively (p <0.001). Pepsin, unconjugated bile salts, and endotoxin were not significantly different in the two groups. Total taurine conjugated bile salts, and specifically taurocholate, was significantly higher in the PPI group (p=0.01 and p=0.005). pH and concentration of deconjugated bile and conjugated bile salts were significantly associated (p=0.006 and p=0.02). Endotoxin and bile salts were not significantly associated. CONCLUSION: Taurine conjugated bile acids are significantly higher in children chronically using PPIs compared to controls. Moreover acidity of gastric pH correlated negatively with deconjugated and conjugated bile salts. These findings imply that GJ under chronic proton pump inhibition contains nonacid components potentially harmful to esophageal mucosa and bronchial tissue.

Su1253 Abnormal Dynamics of Endogenous Acylated Ghrelin Mediates Decreases in Gastric Phase III-Like Contraction, Leading to Delayed Gastric Emptying in Urocortin1-Induced Stress Rats

INTRODUCTION: Gastric acid suppression is justified to prevent severe gastro esophageal reflux (GER) disease related complications. However, it does not reduce the total amount and proximal extent of GER in the esophagus and non-acid components are able to induce (extra-esophageal) GER symptoms as well. No data on the composition of gastric juice (GJ) in children using gastric acid suppression exists. We therefore aimed to assess whether the composition of gastric juice in children using proton pump inhibitors (PPIs) differs, compared to that of their controls. METHODS: Infants and children (0-18 years) on proton pump inhibitors (PPIs) for at least six weeks and a control group not using anti reflux medication, were included. GJ was obtained through an existing nasogastric or a percutaneous endoscopic gastrostomy tube/ Mic-key gastrostomy. In the collected GJ (5 ml), pH, pepsin activity, bile salts and endotoxin (LPS) levels were determined. Pepsin was measured using a fluorometric assay using 4-Methyl-Coumaryl-7-Amide (MCA) substrate with/without pepstatin. Concentrations of deconjugated and taurine/glycine-conjugated bile salts were assessed by reversephase HPLC. Levels of LPS were determined using the spectrophotometric Limulus Amebocyte Lysate assay. RESULTS: GJ was analyzed from 16 children with (median: 3.8 yrs, range: 17.6 years) and 16 children (4.0 yrs, range: 16.0) without PPI therapy. Median duration of PPI treatment was 24 weeks (range: 514 weeks). Gastric pH was 5.0 (range: 5.0) and 1.0 (range: 4.5) in the PPI and control group respectively (p <0.001). Pepsin, unconjugated bile salts, and endotoxin were not significantly different in the two groups. Total taurine conjugated bile salts, and specifically taurocholate, was significantly higher in the PPI group (p=0.01 and p=0.005). pH and concentration of deconjugated bile and conjugated bile salts were significantly associated (p=0.006 and p=0.02). Endotoxin and bile salts were not significantly associated. CONCLUSION: Taurine conjugated bile acids are significantly higher in children chronically using PPIs compared to controls. Moreover acidity of gastric pH correlated negatively with deconjugated and conjugated bile salts. These findings imply that GJ under chronic proton pump inhibition contains nonacid components potentially harmful to esophageal mucosa and bronchial tissue.

The effects of oral administration of bile salt hydrolase on the partition of fatty acid and glucose oxidation in Zucker fatty rats (585.8)

Conjugated bile salts take part in micellar formation, a critical step for dietary lipid digestion and absorption. Bile salt hydrolases (BSHs) catalyze the conversion of conjugated bile salts to bile acids and amino acids. Gut bacteria produce this family of enzymes. BSHs could potentially reduce the efficiency of lipid digestion and absorption if applied to the upper portion of the small intestine. Thus, we hypothesize that BSH is a novel lead for therapeutic reduction of lipid absorption needed to control obesity in humans. To test this, recombinant BSH (rBSH) was expressed and purified from an E. coli expression system by Ni‐NTA affinity chromatography. The rBSH or bovine serum albumin (BSA) control (2.7mg/kg of body weight) was gavaged daily into Zucker fatty (ZF) rats (9‐12 weeks old, 4 per group) just before the start of the dark photoperiod (before active feeding starts) for 7 days. After the last treatment, rats were housed individually in Comprehensive Laboratory Animal Monitoring Systems for 24 hours to collect the indirect calorimetry data. The rats treated with rBSH had significantly higher respiratory exchange ratios (RER) during the dark (p=0.028), but not the light photoperiod. The heat production and change in body weight were not different between groups. The higher RER data of ZF rats treated with rBSH during the dark photoperiod (feeding time) indicate a reduction of energy derived from fat oxidation, suggesting that lipid digestion or absorption may be reduced, which leads to higher utilization of glucose. Together, the findings shown in this pilot study suggest BSH is a lead for novel medication for modulating lipid digestion and absorption.

Preliminary evaluation of probiotic properties of Lactobacillus strains isolated from Sardinian dairy products.

Twenty-three Lactobacillus strains of dairy origin were evaluated for some functional properties relevant to their use as probiotics. A preliminary subtractive screening based on the abilities to inhibit the growth of microbial pathogens and hydrolyze conjugated bile salts was applied, and six strains were selected for further characterization including survival under gastrointestinal environmental conditions, adhesion to gut epithelial tissue, enzymatic activity, and some safety properties. All selected strains maintained elevated cell numbers under conditions simulating passage through the human gastrointestinal tract, well comparable to the values obtained for the probiotic strain Lactobacillus rhamnosus GG, and were able to adhere to Caco-2 cells to various extents (from 3 to 20%). All strains exhibited high aminopeptidase, and absent or very low proteolytic and strong β-galactosidase activities; none was found to be haemolytic or to produce biogenic amines and all were susceptible to tetracycline, chloramphenicol, erythromycin, ampicillin, and amoxicillin/clavulanic acid. Our results indicate that the Lactobacillus strains analyzed could be considered appropriate probiotic candidates, due to resistance to GIT simulated conditions, antimicrobial activity, adhesion to Caco-2 cell-line, and absence of undesirable properties. They could be used as adjunct cultures for contributing to the quality and health related functional properties of dairy products.

Bile Salt Inhibition of Host Cell Damage by Clostridium Difficile Toxins

Virulent Clostridium difficile strains produce toxin A and/or toxin B that are the etiological agents of diarrhea and pseudomembranous colitis. Treatment of C. difficile infections (CDI) has been hampered by resistance to multiple antibiotics, sporulation, emergence of strains with increased virulence, recurrence of the infection, and the lack of drugs that preserve or restore the colonic bacterial flora. As a result, there is new interest in non-antibiotic CDI treatments. The human conjugated bile salt taurocholate was previously shown in our laboratory to inhibit C. difficile toxin A and B activities in an in vitro assay. Here we demonstrate for the first time in an ex vivo assay that taurocholate can protect Caco-2 colonic epithelial cells from the damaging effects of the C. difficile toxins. Using caspase-3 and lactate dehydrogenase assays, we have demonstrated that taurocholate reduced the extent of toxin B-induced apoptosis and cell membrane damage. Confluent Caco-2 cells cultured with toxin B induced elevated caspase-3 activity. Remarkably, addition of 5 mM taurocholate reduced caspase-3 activity in cells treated with 2, 4, 6, and 12 µg/ml of toxin B by 99%, 78%, 64%, and 60%, respectively. Furthermore, spent culture medium from Caco-2 cells incubated with both toxin B and taurocholate exhibited significantly decreased lactate dehydrogenase activity compared to spent culture medium from cells incubated with toxin B only. Our results suggest that the mechanism of taurocholate-mediated inhibition functions at the level of toxin activity since taurocholate did not affect C. difficile growth and toxin production. These findings open up a new avenue for the development of non-antibiotic therapeutics for CDI treatment.

In vitro lipid peroxidation of intestinal bile salt-based nanoemulsions: Potential role of antioxidants

Over the last decades, oxidative stress has been described as a deleterious phenomenon contributing to numerous noncommunicable diseases such as cardiovascular disease, diabetes, and cancers. As many authors ascribed the healthy effect of fruit and vegetable consumption mainly to their antioxidant contents, it has been hypothesized that their protection could occur from the gut. Therefore, the aim of this study was to develop an original and physiological model of nanoemulsions to study lipid peroxidation within the intestine and to assess the properties of potential antioxidants in this setting.Several nanoemulsions were compared in terms of physical characteristics and reactivity to 2,2’-azobis-(2-amidinopropane) hydrochloride (AAPH)-induced oxidation. Formulations included different types of lipids, a detergent (a conjugated bile salt or sodium dodecyl sulfate) and, finally, lipophilic antioxidants. Hemin and myoglobin were also tested as relevant potential oxidants. Fatty acid (FA) peroxidation was monitored by gas chromatography while malondialdehyde and antioxidant contents were measured by HPLC.Investigated nanoemulsions were composed of spherical or cylindrical mixed micelles, the latter being the least resistant to oxidation. In the experimental conditions, AAPH was the only efficient oxidant. Alpha-tocopherol and lutein significantly slowed FA degradation from 4 to 1 μM, respectively. On the contrary, beta-carotene did not show any protective capacity at 4 μM.In conclusion, the tested nanoemulsions were appropriate to assess antioxidant capacity during the intestinal phase of digestion.

Studies on the Action Mechanism for Cholesterol-Lowering of &lt;i&gt;Lactobacillus&lt;/i&gt; which Yields Bile Salt Hydrolase from Kefir Grains

Cholesterol-lowering strains were obtained by high throughput screening technology and ortho-phthalaldehyde method. We used oxford cup method to screen again to obtain strains of high yield bile salt hydrolase and illuminate action mechanism ofLactobacillusreducing cholesterol. Screened six strains had the ability of high yield bile salt hydrolase and good ferment ability. The results of identifying bacteria species: strain KTxKL1J1 wereLactobacillus casei, strain Tx wasStreptococcus thermophilus, strain KS4P1 wereLactococcus lactis subsp.lactis, where the last two bacteria were strain of high yield bile salt hydrolase to be few known in literature. This work showed that dissociation bile salts and cholesterol conjuncted sediments by bile salt hydrolase decomposing conjugated bile salts.

Bile resistance mechanisms in Lactobacillus and Bifidobacterium

Probiotics are live microorganisms which when administered in adequate amounts confer a health benefit on the host. Most of the probiotic bacteria currently available in the market belong to the genera Lactobacillus and Bifidobacterium, and specific health-promoting activities, such as treatment of diarrhea or amelioration of gastrointestinal discomfort, have been attributed to them. In order to be able to survive the gastrointestinal transit and transiently colonize our gut, these bacteria must be able to counteract the deleterious action of bile salts, which are the main components of bile. Bile salts are detergent-like biological substances synthesized in the liver from cholesterol. Host enzymes conjugate the newly synthesized free bile acids in the liver with the amino acids glycine or taurine, generating conjugated bile salts. These compounds are stored in the gall bladder and they are released into the duodenum during digestion to perform their physiological function, which is the solubilization of fat coming from diet. These bile salts possess strong antimicrobial activity, since they are able to disorganize the structure of the cell membrane, as well as trigger DNA damage. This means that bacteria inhabiting our intestinal tract must have intrinsic resistance mechanisms to cope with bile salts. To do that, Lactobacillus and Bifidobacterium display a variety of proteins devoted to the efflux of bile salts or protons, to modify sugar metabolism or to prevent protein misfolding. In this manuscript, we review and discuss specific bile resistance mechanisms, as well as the processes responsible for the adaptation of bifidobacteria and lactobacilli to bile.

Phase 1 and Phase 2 Drug Metabolism and Bile Acid Production of HepaRG Cells in a Bioartificial Liver in Absence of Dimethyl Sulfoxide

The human liver cell line HepaRG has been recognized as a promising source for in vitro testing of metabolism and toxicity of compounds. However, currently the hepatic differentiation of these cells relies on exposure to dimethylsulfoxide (DMSO), which, as a side effect, has a cytotoxic effect and represses an all-round hepatic functionality. The AMC-bioartificial liver (AMC-BAL) is a three-dimensional bioreactor that has previously been shown to upregulate various liver functions of cultured cells. We therefore cultured HepaRG cells in the AMC-BAL without DMSO and characterized the drug metabolism. Within 14 days of culture, the HepaRG-AMC-BALs contained highly polarized viable liver-like tissue with heterogeneous expression of CYP3A4. We found a substantial metabolism of the tested substrates, ranging from 26% (UDP-glucuronosyltransferase 1A1), 47% (CYP3A4), to 240% (CYP2C9) of primary human hepatocytes. The CYP3A4 activity could be induced 2-fold by rifampicin, whereas CYP2C9 activity remained equally high. The HepaRG-AMC-BAL secreted bile acids at 43% the rate of primary human hepatocytes and demonstrated hydroxylation, conjugation, and transport of bile salts. Concluding, culturing HepaRG cells in the AMC-BAL yields substantial phase 1 and phase 2 drug metabolism, while maintaining high viability, rendering DMSO addition superfluous for the promotion of drug metabolism. Therefore, AMC-BAL culturing makes the HepaRG cells more suitable for testing metabolism and toxicity of drugs.

The Vesicular Transport Assay: Validated In Vitro Methods to Study Drug‐Mediated Inhibition of Canalicular Efflux Transporters ABCB11/BSEP and ABCC2/MRP2

The canalicular membrane of hepatocytes contains several transport proteins that use the energy of ATP to efflux potentially toxic molecules to the bile. Probably the two most important proteins at this location are MRP2 and BSEP, which transport phase II conjugates of xenobiotics and endobiotics and conjugated bile salts, respectively. The impaired function of either of these transporter proteins reduces the clearance of the toxic conjugates, resulting in their accumulation in the hepatocytes and eventually the plasma. Conjugated bile salts and phase II metabolites are compounds with low passive permeability; therefore, the most commonly used test system to investigate MRP2- and BSEP-mediated transport processes is the vesicular transport assay. The concentration of probe substrates and inhibitors used in the experiment is close to their free concentration in the hepatocytes, providing an advantage when calculating kinetic parameters (K(m), K(i), V(max)). The protocols aim to assist scientists to set up a transport assay for a known or potential substrate and test small molecule inhibition of the transporters.

Mitigating the Inhibition of Human Bile Salt Export Pump by Drugs: Opportunities Provided by Physicochemical Property Modulation, In Silico Modeling, and Structural Modification

The human bile salt export pump (BSEP) is a membrane protein expressed on the canalicular plasma membrane domain of hepatocytes, which mediates active transport of unconjugated and conjugated bile salts from liver cells into bile. BSEP activity therefore plays an important role in bile flow. In humans, genetically inherited defects in BSEP expression or activity cause cholestatic liver injury, and many drugs that cause cholestatic drug-induced liver injury (DILI) in humans have been shown to inhibit BSEP activity in vitro and in vivo. These findings suggest that inhibition of BSEP activity by drugs could be one of the mechanisms that initiate human DILI. To gain insight into the chemical features responsible for BSEP inhibition, we have used a recently described in vitro membrane vesicle BSEP inhibition assay to quantify transporter inhibition for a set of 624 compounds. The relationship between BSEP inhibition and molecular physicochemical properties was investigated, and our results show that lipophilicity and molecular size are significantly correlated with BSEP inhibition. This data set was further used to build predictive BSEP classification models through multiple quantitative structure-activity relationship modeling approaches. The highest level of predictive accuracy was provided by a support vector machine model (accuracy = 0.87, κ = 0.74). These analyses highlight the potential value that can be gained by combining computational methods with experimental efforts in early stages of drug discovery projects to minimize the propensity of drug candidates to inhibit BSEP.

Brucella abortus Choloylglycine Hydrolase Affects Cell Envelope Composition and Host Cell Internalization

Choloylglycine hydrolase (CGH, E.C. 3.5.1.24) is a conjugated bile salt hydrolase that catalyses the hydrolysis of the amide bond in conjugated bile acids. Bile salt hydrolases are expressed by gastrointestinal bacteria, and they presumably decrease the toxicity of host's conjugated bile salts. Brucella species are the causative agents of brucellosis, a disease affecting livestock and humans. CGH confers Brucella the ability to deconjugate and resist the antimicrobial action of bile salts, contributing to the establishment of a successful infection through the oral route in mice. Additionally, cgh-deletion mutant was also attenuated in intraperitoneally inoculated mice, which suggests that CGH may play a role during systemic infection other than hydrolyzing conjugated bile acids. To understand the role CGH plays in B. abortus virulence, we infected phagocytic and epithelial cells with a cgh-deletion mutant (Δcgh) and found that it is defective in the internalization process. This defect along with the increased resistance of Δcgh to the antimicrobial action of polymyxin B, prompted an analysis of the cell envelope of this mutant. Two-dimensional electrophoretic profiles of Δcgh cell envelope-associated proteins showed an altered expression of Omp2b and different members of the Omp25/31 family. These results were confirmed by Western blot analysis with monoclonal antibodies. Altogether, the results indicate that Brucella CGH not only participates in deconjugation of bile salts but also affects overall membrane composition and host cell internalization.