Bile Salt Treatment Research Articles

Acquiring bifidobacteria species from formula-fed and breast-fed newborns: identifying, quantifying and creating an antibiogram.

After examining the Bifidobacterium spp. population in faeces samples from breast-fed and formula-fed infants, an antibiogram was created. The prevalence of Bifidobacterium spp. in faeces was determined using common bacterial growth media, including Man Rogos Sharpe (MRS), Brain Heart Infusion (BHI), Luria Bertani (LB) broth and Bifidobacteria agar. According to the findings, formula-fed babies had a low population of Bifidobacterium spp. in their stools while breast-fed babies had a high population. By using phylogenetic analysis of the 16S rRNA and xfp (xylose/fructose 6-phosphate phosphoketolase) genes, and RFLP mapping of Bifidobacterium isolates, it was possible to identify a new and unique Bifidobacterium species. The intensity of the reddish brown colour produced during the F6PPK (fructose 6-phosphate phosphoketolase) assay is an accurate indicator of the proportion of various bifidobacteria present. Bifidobacteria agar media produced the greatest amounts of bifidobacteria diversity and recovery. Small (SCV) and Big colony variations (BCV) were formed during growth on different media. The various antibiotic MIC values changed depending on the use of different media, growth circumstances, bile salt treatment and low pH. The findings of this study demonstrate that test conditions also impact the diversity of microbiological conditions that distinguish between resistant and susceptible bacteria.

Isolation, characterization and safety assessment of probiotic lactic acid bacteria from metata ayib (Traditional spiced cottage cheese)

BackgroundConsumption of fermented foods and beverages has health benefits; fermented milk products in particular are thought to contain probiotics. The purpose of this study was to characterize probiotic property of lactic acid bacteria isolates from metata ayib. MethodsStandard method with MRS agar was used to isolate and purify LAB isolates from metata ayib. Purified LAB colinies were characterized for its probiotic property using tests for tolerance at low pH (3.0, 2.5, and 2.0), bile salt (0.5% and 0.3%), adherence (Congo red), antagonistic activity, and antibiotic susceptibility. Finally, morphological and physiological tests were used to identify LAB colonies with putative probiotic properties. ResultsA total of 80 LAB isolates were isolated and purified. The majority of isolates, 67 (83.75%), had bacilli shapes, and 59 (73.75%) were homofermentative. All 80 (100%) isolates were tolerated at pH 3.0 treated for three hours; but only 6 (7.5%) isolates were survived at pH 2.0. After 0.3% and 0.5% bile salt treatment for 24 h, all 6 (7.5%) LAB isolates survived above 92.09 ± 0.18 and 83.39 ± 0.21%, respectively. All the 6 isolates were identified as Lactobacillus species; and all were hydrophobicity positive. K. pneumoniae was resistant but S. aureus was sensitive to all of six identified LAB isolates cell free supernatant. All 6 isolates were ciprofloxacin, chloramphenicol, cotrimoxazole and erythromycin-sensitive. Vancomycin resistance was present in four isolates. All these six Lactobacilli species were non-hemolytic, non-biogenic amine producing, and gelatin hydrolysis negative. ConclusionThus, Lactobacilli species isolated from metata ayib may be used as the suitable candidates of probiotics, and bio-therapeutics agents against bacterial infection to humans.

P53 Deficiency Accelerate Esophageal Epithelium Intestinal Metaplasia Malignancy

Barrett's esophagus (BE) is a precancerous lesion of esophageal adenocarcinoma (EAC). It is a pathological change in which the squamous epithelium distal esophagus is replaced by columnar epithelium. Loss of P53 is involved in the development of BE and is taken as a risk factor for the progression. We established a HET1A cell line with P53 stably knockdown by adenovirus vector infection, followed by 30 days of successive acidic bile salt treatment. MTT, transwell assay, and wound closure assay were applied to assess cell proliferation and migration ability. The expression of key factors was analyzed by RT-qPCR, western blotting and immunohistochemical staining. Our data show that the protein expression level of P53 reduced after exposure to acidic bile salt treatment, and the P53 deficiency favors the survival of esophageal epithelial cells to accommodate the stimulation of acidic bile salts. Furthermore, exposure to acidic bile salt decreases cell adhesions by repressing the JAK/STAT signaling pathway and activating VEGFR/AKT in P53-deficient esophageal cells. In EAC clinical samples, P53 protein expression is positively correlated with that of ICAM1 and STAT3 and negatively correlated with VEGFR protein expression levels. These findings elucidate the role of P53 in the formation of BE, explain the mechanism of P53 deficiency as a higher risk of progression for BE formation, and provide potential therapeutic targets for EAC.

Identification of Novel Bile Salt-Tolerant Genes in Lactobacillus Using Comparative Genomics and Its Application in the Rapid Screening of Tolerant Strains.

Under bile salt treatment, strains display significant differences in their tolerance ability, suggesting the existence of diverse resistance mechanisms in Lactobacillus; however, the genes involved in this protective process are not fully understood. In this study, novel target genes associated with bile salt tolerance in Lactobacillus were identified using comparative genomics for PCR detection and the rapid screening of tolerant strains. The bile salt tolerance of 107 lactobacilli isolated from different origins was assessed, and 26 strains with comparatively large differences were selected for further comparative genomic analysis. Tolerant strains had 112 specific genes that were enriched in the phosphotransferase system, the two-component system, carbohydrate metabolism, and the ATP-binding cassette transporter. Six genes from Lactobacillus were cloned into the inducible lactobacillal expression vector pSIP403. Overexpression in the host strain increased its tolerance ability by 11.86-18.08%. The novel genes identified here can be used as targets to design primers for the rapid screening of bile salt-tolerant lactobacilli. Altogether, these results deepen our understanding of bile salt tolerance mechanisms in Lactobacillus and provide a basis for further rapid assessments of tolerant strains.

Microbial and physicochemical properties of spray dried kefir microcapsules during storage

To preserve the probiotic and functional potential of kefir and increase its stability, this study aimed to characterize and evaluate the effect of microencapsulation on the viability and stability of kefir microparticles storage and simulated gastrointestinal conditions. Kefir microencapsulation was performed using the spray drying with different encapsulating matrices and without a coating material at 140 °C. Seven organic acids were identified and quantified, with lactic acid found in the highest amounts. Different wall material influenced the hygroscopicity, dispersibility and solubility stability. Fresh kefir had 9.64 and 7.60 log CFU·g−1 lactic acid bacteria (LAB) and yeast counts, respectively. After the spray drying at 140 °C, all samples at 4 °C had lower LAB counts. The use of MD (p = 0.7819) or CAP (p = 0.0507) affected positively the stability of LAB, regardless of storage temperature over 77 days. Yeast counting has higher at 4 °C in microparticles with or without wall encapsulant. Lactobacillus lactis and Lactococcus mesenteroides survived the acid pH and bile salt treatment. Kefir beverage had a high overall acceptance by non-trained judges compared to fresh kefir. In conclusion, kefir encapsulation by spray-dryer allowed developing a fermented food product preserved, stable and source of bioactive compounds.

Synergism of Dietary Co-Supplementation with Lutein and Bile Salts Improved the Growth Performance, Carotenoid Content, Antioxidant Capacity, Lipid Metabolism, and Lipase Activity of the Marbled Spinefoot Rabbitfish, Siganus rivulatus.

Simple SummaryIn the natural aquatic environment, fish obtain their carotenoid requirements from the natural food web. Meanwhile, under aquaculture conditions, especially intensive culture, fish are deprived in terms of their carotenoids requirements from the environment. Accordingly, the artificial diet formula must consider the carotenoids accessibility and maintain its efficient utilization. Moreover, in fish larvae, the absorption of carotenoids as lutein (LTN) is not efficient. Therefore, as LTN is fat-soluble carotenoid, the improvement of fat digestion and absorption could improve LTN utilization. The present work evaluated the effect of individual or co-supplementation of LTN and bile salts (BS) in the diets of rabbitfish (Siganus rivulatus) larvae. The results revealed an improvement of growth performance and survival (%) with dietary supplementation with LTN and BS, which could enhance the cost–benefit of farming rabbitfish. Also, the carotenoid deposition, antioxidant status, lipase activity, and lipid metabolism improved with co-supplementation of LTN and BS than its individual supplementation.A 60-day feeding trial was conducted to assess the effects of dietary supplementation with bile salts (BS), lutein (LTN), and their combination on growth, survival, carotenoid content, and antioxidant status of rabbitfish (Siganus rivulatus) larvae. Fish were fed four isonitrogenous (34.39% protein) and isoenergetic (20.57 kJ/g) diets supplemented with BS (0.15 g kg−1), LTN (0.1 g kg−1), BS+LTN (0.15 and 0.1 g kg−1, respectively), and a non-supplemented control diet. The results revealed that fish fed BS+LTN had the highest significant specific growth rate (4.37% day−1), feed efficiency (46.55%), and survival (97.78%). Lutein supplementation improved whole-body protein content, meanwhile, fish fed a BS-supplemented diet had a higher lipid content. The carotenoid deposition was significantly increased with LTN and BS+LTN in skin, muscle, and whole body compared to the control and BS treatment. All dietary supplementation of BS and LTN showed significant improvement in total antioxidant capacity, catalase, and glutathione peroxidase activities. Additionally, LTN alone or BS+LTN significantly reduced malondialdehyde levels by 5.30 and 29.91%, respectively compared to the control. BS supplementation modulated aminopeptidases activities, triglycerides, cholesterol, and increased the activity of pancreatic lipase. Therefore, it could be inferred that dietary supplementation with LTN in combination with BS could improve the growth performance, carotenoid deposition, antioxidant status, lipid digestion, and metabolism of S. rivulatus.

Abstract 878: IGFBP2 is required to activate EGFR-DNA-PKcs pathway to protect esophageal adenocarcinoma cells from acidic bile salts-induced DNA damage

Abstract The incidence of esophageal adenocarcinoma (EAC) is rising rapidly in the US and Western countries. The development of Barrett’s esophagus (BE) and its progression to EAC have been linked to gastroesophageal reflux disease (GERD). Exposure of BE and EAC cells to acidic bile salts (ABS) in GERD conditions induces high levels of DNA damage. In this study, we investigated the role of insulin-like growth factor binding protein 2 (IGFBP2) in regulating ABS-induced DNA double-strand breaks (DSBs). Immunohistochemistry analysis demonstrated frequent overexpression of IGFBP2 in EACs (31/57). Treatment of EAC cells with ABS, to mimic GERD conditions, induced high levels of IGFBP2 expression. Knocking down endogenous IGFBP2 in FLO1 cells (with constitutive high levels of IGFBP2) led to a significant increase in DNA DSBs and apoptosis, following transient exposure (20 min) to ABS. On the other hand, overexpression of exogenous IGFBP2 in OE33 cells (with low endogenous levels of IGFBP2) had a protective effect against DSBs and apoptosis. Using western blotting and immunofluorescence assays, we found that IGFBP2 is required for ABS-induced nuclear accumulation and phosphorylation of EGFR and DNA-PKcs, which are required for DNA damage repair activity. Using co-immunoprecipitation assay, we detected IGFBP2 forms complex with EGFR and DNA-PKcs and co-localizes with EGFR and DNA-PKcs in nucleus, following acidic bile salts treatment. We also demonstrated, using cycloheximide (CHX) chase assay, that IGFBP2 promotes EGFR protein stability in response to ABS exposure. In summary, our data indicate that IGFBP2 protects EAC cells against ABS-induced DNA damage and apoptosis through stabilization and activation of EGFR - DNA-PKcs signaling axis. Citation Format: Zhangjian Zhou, Heng Lu, Shoumin Zhu, Ahmed Gomaa, Zheng Chen, Jin Yan, Wael El-Rifai, Chengxue Dang, Dunfa Peng. IGFBP2 is required to activate EGFR-DNA-PKcs pathway to protect esophageal adenocarcinoma cells from acidic bile salts-induced DNA damage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 878.

Activation of EGFR-DNA-PKcs pathway by IGFBP2 protects esophageal adenocarcinoma cells from acidic bile salts-induced DNA damage

BackgroundThe incidence of esophageal adenocarcinoma (EAC) is rising rapidly in the US and Western countries. The development of Barrett’s esophagus (BE) and its progression to EAC have been linked to chronic gastroesophageal reflux disease (GERD). Exposure of BE and EAC cells to acidic bile salts (ABS) in GERD conditions induces high levels of oxidative stress and DNA damage. In this study, we investigated the role of insulin-like growth factor binding protein 2 (IGFBP2) in regulating ABS-induced DNA double-strand breaks.MethodsReal-time RT-PCR, western blot, immunohistochemistry, immunofluorescence, co-immunoprecipitation, flow cytometry, and cycloheximide (CHX) chase assays were used in this study. To mimic GERD conditions, a cocktail of acidic bile salts (pH 4) was used in 2D and 3D organotypic culture models. Overexpression and knockdown of IGFBP2 in EAC cells were established to examine the functional and mechanistic roles of IGFBP2 in ABS-induced DNA damage.ResultsOur results demonstrated high levels of IGFBP2 mRNA and protein in EAC cell lines as compared to precancerous Barrett’s cell lines, and IGFBP2 is frequently overexpressed in EACs (31/57). Treatment of EAC cells with ABS, to mimic GERD conditions, induced high levels of IGFBP2 expression. Knocking down endogenous IGFBP2 in FLO1 cells (with constitutive high levels of IGFBP2) led to a significant increase in DNA double-strand breaks and apoptosis, following transient exposure to ABS. On the other hand, overexpression of exogenous IGFBP2 in OE33 cells (with low endogenous levels of IGFBP2) had a protective effect against ABS-induced double-strand breaks and apoptosis. We found that IGFBP2 is required for ABS-induced nuclear accumulation and phosphorylation of EGFR and DNA-PKcs, which are necessary for DNA damage repair activity. Using co-immunoprecipitation assay, we detected co-localization of IGFBP2 with EGFR and DNA-PKcs, following acidic bile salts treatment. We further demonstrated, using cycloheximide chase assay, that IGFBP2 promotes EGFR protein stability in response to ABS exposure.ConclusionsIGFBP2 protects EAC cells against ABS-induced DNA damage and apoptosis through stabilization and activation of EGFR - DNA-PKcs signaling axis.

Exposure of Barrett's and esophageal adenocarcinoma cells to bile acids activates EGFR-STAT3 signaling axis via induction of APE1.

The development of Barret’s esophagus (BE) and its progression to esophageal adenocarcinoma (EAC) is highly linked to exposure to acidic bile salts due to chronic gastroesophageal reflux disease (GERD). In this study, we investigated the role of Apurinic/apyrimidinic endonuclease 1 /redox effector factor-1 (APE-1/REF-1) in STAT3 activation in response to EAC. Our results indicate that APE1 is constitutively overexpressed in EAC whereas its expression is transiently induced in response to acidic bile salts in non-neoplastic BE. Using overexpression or shRNA knockdown of APE1, we found that APE1 is required for phosphorylation, nuclear localization, and transcription activation of STAT3. By using an APE1 redox-specific mutant (C65A) and APE1 redox inhibitor (E3330), we demonstrate that APE1 activates STAT3 in a redox-dependent manner. By using pharmacologic inhibitors and genetic knockdown systems, we found that EGFR is a required link between APE1 and STAT3. EGFR phosphorylation (Y1068) was directly associated with APE1 levels and redox function. Co-immunoprecipitation and proximity ligation assays indicated that APE-1 coexists and interacts with the EGFR-STAT3 protein complex. Consistent with these findings, we demonstrated a significant induction in mRNA expression levels of STAT3 target genes (IL-6, IL-17A, BCL-xL, Survivin and c-Myc) in BE and EAC cells, following acidic bile salts treatment. ChIP assays indicated that acidic bile salts treatment enhances binding of STAT3 to the promoter of its target genes, Survivin and BCL-xL. Inhibition of APE1/REF-1 redox activity using E3330 abrogated STAT3 DNA binding and transcriptional activity. The induction of APE-1 - STAT3 axis in acidic bile salts conditions provided a survival advantage and promoted cellular proliferation. In summary, our study provides multiple pieces of evidence supporting a critical role for APE1 induction in activating the EGFR-STAT3 signaling axis in response to acidic bile salts, the main risk factors for Barrett’s carcinogenesis.

Characterization of extracellular yeast peptide factors and their stress-protective effect on probiotic lactic acid bacteria

Protective effect of the extracellular peptide fraction (reactivating factors, RF) produced by yeasts of various taxonomic groups (Saccharomyces cerevisiae, Kluyveromyces lactis, Candida utilis, and Yarrowia li- polytica) on probiotic lactic acid bacteria (LAB) Lactobacillus casei, L. acidophilus,'and L. reuteri under bile salt (BS)-induced stress was shown. RF of all yeasts were shown to be of peptide nature; the active component of the S. cerevisiae RF was identified as a combination of low-molecular polypeptides with molecular masses of 0.6 to 1.5 kDa. The protective and reactivating effects of the yeast factors were not species-specific and were similar to those of the Luteococcusjaponicus subsp. casei R. In BS-treated cells of the tester bacteria, a pro- tective effect was observed after 10-min preincubation of the LAB cell suspension with yeast RE: the number of surviving cells (CFU) was 2 to 4.5 times higher than in the control. The reactivating effect was observed when RF was added to LAB cell suspensions not later than 15 min after stress treatment. It was less pro- nounced than the protector effect, with the CFU number I to 3 times that of the control. Both the protector and the reactivating effects were most pronounced in the S. cerevisiae and decreased in the row: C. utilis > K. lactis > Y lipolytica. The efficiency of protective action of yeast RF was found to depend on the properties of recepient LAB cells, with the L. casei strain being most sensitive to BS treatment. In both variants, the highest protective effect of RF (increase in the CFU number) was observed for L. acidophilus, while the least pronounced one, for L. casei. The reasons for application of the LAB strains combining high stress resistance and high response to stress-protecting metabolites, including RF factors, as probiotics, is discussed.

Structural characterization of the extracellular peptide metabolites of Luteococcus japonicus subsp. casei and their protective effect on probiotic bacteria

Protective effect of Luteococcus japonicus subsp. casei exometabolites on the cells of transient probiotic Propionibacterium strains, Enterococcus faecium, and the yeasts Saccharomyces cerevisiae Boulardii under exposure to bile salts (BS) and acid stress was studied. The extracellular peptide reactivating factor (RF) and the peptide component of the culture liquid (CL) after RF removal possessed a protective effect. Protective (preventive) and reactivating (after stress impact) application of RF resulted in 1.5- to 2.0-fold increased survival of the human transient probiotics P. freudenreichii, P. acidipropionici and E. faecium subjected to BS treatment of acid stress. The CL peptide fraction had a stronger protective effect. Its application for preincubation of P. acidipropionici cells resulted in 14-fold (BS treatment) and 8-fold (acid stress) increased survival compared to the control. Yeasts exhibited very high resistance to the stress factors used. Two active glycopeptide fractions with molecular masses of 1.8 and 2.4 kDa were found in RF. Analysis of their amino acid composition revealed the residues of glycine, leucine, proline, arginine, and aspartate/asparagine, while no residues of aromatic and sulfur-containing amino acids, or the disulfide bridge-type posttranslational modifications, were found. The possible mechanisms of reactivating and the protective effect of RF are discussed.

Preliminary evaluation of probiotic potential of Lactobacillus plantarum strains isolated from Italian food products

The aim of this study was to investigate some probiotic properties of 42 wild Lactobacillus plantarum strains isolated from different Italian foods of animal origin. The strains were first screened for their antibiotic resistance profile (chloramphenicol, erythromycin, gentamicin, and tetracycline), subsequently they were tested for their in vitro resistance to lysozyme (100 mg L⁻¹), low pH (3.0, 2.5 and 2.0) and bile salts (0.3, 0.5 and 1.0 %). Moreover, agglutination property was studied (adhesion to Saccharomyces cerevisiae cells), as well as the presence of bsh and msa genes. The strains with the best characteristics were subjected to a further trial in order to evaluate their ability to survive to multiple stresses over time (lysozyme, low pH and bile salts) and the effect of these treatments on adhesion to yeast cells. All the strains were susceptible to chloramphenicol, erythromycin and gentamicin, while 6 strains were excluded from further evaluation because of their resistant phenotype against tetracycline. All the strains were able to grow in presence of lysozyme, as well as in MRS broth at pH 3.0. Only 4 strains showed a growth rate lower than 80 % when grown in MRS broth at pH 2.5, while a relevant growth rate decrease was observed after exposure to pH 2.0. Bile salts didn't affect the viability of the L. plantarum cells. Twenty-one strains out of 33 tested strains were able to adhere to S. cerevisiae cells. Presence of both bsh and msa genes was detected in 6 strains. The strains resistant to all the stresses, positive to agglutination with S. cerevisiae and showing bsh and msa genes were selected for further evaluation and subjected to different stress treatments over time. The assessment of growth rates showed that exposure to lysozyme significantly increased low pH resistance in L. plantarum. This increase ranged from 2.35 to 15.57 %. The consequential lysozyme and low pH exposures didn't affect the growth rate values after bile salts treatment, as well as the ability of the strains to adhere to yeast cells wasn't modified by previous treatments (lysozyme, low pH and bile salts). The present work allows to increase knowledge about non starter lactic acid bacteria from Italian food products. The studied L. plantarum strains showed a good potential for their use as probiotic cultures. However, more in vivo tests are necessary to confirm this potentiality.

Identification and characterization of WhiB-like family proteins of the Bifidobacterium genus

Bifidobacteria are strictly anaerobic bacteria, that are an important component of human microbiote due to their probiotic characteristics. They are frequently exposed to a variety of stresses, therefore, identification of genes implicated in stress responses in bifidobacteria is critical for biomedicine and maintenance of industrial strains. The WhiB-like family proteins unique for Actinobacteria are transcriptional regulators involved in major cellular processes, including stress responses. The aim of this study was the identification of WhiB-like family proteins of the Bifidobacterium genus of the Actinobacteria class and functional characterization of conservative whiB-like genes. The DNA sequence database of 36 strains revealed a family of WhiB-encoding genes. It were identified the wblE orthologs in all Bifidobacteria species and the whiB2 orthologs in all bifidobacterial strains except of all strains of Bifidobacterium animalis subsp. lactis and Bifidobacterium gallicum. Some strains, in particular, those of the Bifidobacterium longum group, contain additional whiB-like genes of different length and a low degree of similarity in sequences. The wblE and whiB2 genes of the Bifidobacterium genus are evolutionary conservative and ancient genes. The real-time PCR analysis showed that transcription of wblE is induced by a variety of stress conditions such as heat shock, osmotic, oxidative stresses, by antibiotic tetracycline and bile salt treatment, the nutrient starvation and entry into late stationary phase. The wblE gene may play a significant role in general stress responses in bifidobacteria.

Survival of lactic acid bacteria from fermented milks in an in vitro digestion model exploiting sequential incubation in human gastric and duodenum juice

In the present study, the survival of 9 lactic acid bacteria (5 Lactococcus strains, 3 Lactobacillus strains, and 1 strain of Enterococcus hirae), was investigated in vitro under conditions similar to human digestion using human gastric and duodenal juices. The tolerance of the bacteria was also tested with traditional methods using acidic conditions and bile salts. The strains were subjected to a model digestive system comprising sequential incubation in human gastric and duodenal juices, in a 2-step digestion assay at 37°C, simulating the human upper gastrointestinal tract with human gastric juices at pH 2.5 and human duodenal juices at pH 7. The bacterial strains were tested either as washed cells from culture media or in fermented milk. The initial in vitro testing in acid and bile salts showed that Lactobacillus strains and the E. hirae strain displayed a significantly higher acid tolerance than the lactococci. The lactobacilli and the Enterococcus numbers increased, whereas the lactococci decreased at least 1 log during the bile salt treatment. The Lactobacillus strains showed the highest survival rate in the model digestive system when washed bacterial cultures were used with a minor log reduction, whereas the lactococci numbers were reduced by at least log 4. However, when using fermented milks in the model digestion system it was demonstrated that the Enterococcus strain and 2 strains of Lactococcus lactis ssp. cremoris benefited significantly from the presence of the fermented milk as food matrix, with log numbers >log 7 and 5, respectively, after digestion of the fermented milk. The analyses reported comprise a comprehensive in vitro testing regimen suitable for evaluation of the survival of candidate probiotic bacteria in human digestion as an initial prescreen to clinical trials.

Bile Salts Induce Resistance to Polymyxin in Enterohemorrhagic Escherichia coliO157:H7

Many enteric bacteria use bile as an environmental cue to signal resistance and virulence gene expression. Microarray analysis of enterohemorrhagic Escherichia coli O157:H7 (EHEC) treated with bile salts revealed upregulation of genes for an efflux system (acrAB), a two-component signal transduction system (basRS/pmrAB), and lipid A modification (arnBCADTEF and ugd). Bile salt treatment of EHEC produced a basS- and arnT-dependent resistance to polymyxin.

Colocalization of MnSOD expression in response to oxidative stress

The loss of manganese superoxide dismutase function has been associated with increased incidence of Barrett's esophagus and esophageal adenocarcinoma. In previous studies, we have demonstrated that loss of MnSOD resulted in severe esophageal damage by both endogenous and exogenous bile. However, the alterative manner of MnSOD in esophageal epithelium is largely unknown. In this study, we investigated the expression and localization of MnSOD in response to the exposure to bile salts in an esophageal epithelial cell line. Het-1A cells were seeded at 5 x 10(5) and 10(7) and incubated with taurocholate, cholate, glycocholate, deoxycholate, and the mixture of these bile salts. Mitochondria and cytoplasma were separated, and the expression and localization of MnSOD was determined by Western blot and immunocytochemical assay. Proliferation rates were strongly inhibited in the groups with taurocholate and bile salts mixture at 4 h, with 0.367 +/- 0.042 and 0.396 +/- 0.046, respectively, compared to 0.684 +/- 0.054 in untreated groups (P < 0.05). An increased apoptotic rate compared to untreated group (3.65 +/- 0.59) were significantly increased in taurocholate group and in bile salts mixture group were 33.62 +/- 10.25 and 31.52 +/- 8.97 at 4 h, respectively (P < 0.05). The protein level of MnSOD in mitochondria was increased at 4 h, but with a decreased enzymatic activity after bile salts treatment. Cytoplasmic MnSOD was detected in the cells with bile salts treatment. Immunocytochemical staining demonstrated that esophageal epithelial cell underwent morphological alteration and MnSOD relocalization after bile salts treatment. This is the first study to demonstrate cellular cytosolic MnSOD expression and that this relocalization to the cytosol is a cause for decreased MnSOD enzymatic activity. This suggests that bile salts may contribute to the dysfunction of mitochondria, by enzymatically inhibiting of MnSOD localization and thus activation in the mitochondria.

Effective Treatment of Unconjugated Hyperbilirubinemia With Oral Bile Salts in Gunn Rats

We tested the hypothesis that oral administration of bile salts, which are known to increase the biliary excretion of unconjugated bilirubin (UCB), decreases unconjugated hyperbilirubinemia in the Gunn rat model. Adult Gunn rats were fed a standard diet or the same diet supplemented with 0.5 weight % ursodeoxycholic acid (UDCA) or cholic acid (CA) for 1 or 6 weeks. UCB and urobilinoids, a family of intestinal UCB breakdown products, were determined in plasma, feces, or both. After 6 weeks of treatment, tracer 3H-UCB was administered intravenously to determine steady-state UCB kinetics over the next 60 hours. One-week treatment with UDCA or CA decreased plasma UCB concentrations by 21% and 30%, respectively (each P < .01). During the first 4 days of treatment, both UDCA and CA increased the combined fecal excretion of UCB and urobilinoids (+52% and +32%, respectively; each P < .01). Prolongation of treatment to 6 weeks caused a persistent decrease in plasma UCB concentrations to approximately 40% below baseline (each bile salt P < .001). (3)H-UCB kinetic studies showed that UDCA and CA administration decreased UCB pool size (-33% and -32%, respectively; each P < .05) and increased UCB fractional turnover (+33% and +25%, respectively; each P < .05). Dietary bile salt administration induces a large, persistent decrease in plasma UCB concentrations in Gunn rats. Both UDCA and CA enhance UCB turnover by increasing its fecal disposal. These results support the application of oral bile salt treatment in patients with unconjugated hyperbilirubinemia.

Effect of heat-shock and bile salts on protein synthesis of Bifidobacterium longum revealed by [ 35S]methionine labelling and two-dimensional gel electrophoresis

Experimental conditions for efficient protein radiolabelling and two-dimensional gel electrophoresis were developed for Bifidobacterium longum. Using these tools, protein synthesis in cells before and after heat-shock and bile salts treatment was investigated. Following heat-stress, 13 proteins were upregulated, of which HtrA, DnaK and GroEL were also moderately induced by bile salts, indicating close relationship between the heat and bile salts responses in bifidobacteria. Our work indicated that, as a consequence of prolonged heat-stress, HtrA undergoes sequential modification and proteolysis, and that this mechanism could be employed by bifidobacteria to respond to heat-stress.

Physiological and molecular aspects of bile salt response in Enterococcus faecalis

Analysis of the susceptibility and the acquisition of tolerance in Enterococus faecalis towards bile salts showed a nearly instantaneous killing effect and yielded evidence for homologous tolerance as well as for cross-protections. Two-dimensional (2-D) electrophoresis revealed 45 proteins which are amplified in response to the bile salt treatment. These include a set of seven proteins, the synthesis of which is increased not only with the bile salts but also with multiple sublethal stresses of various nature. Characterisation of the latter (called general stress proteins) showed that at least five of them are related to resistance to bile salts, heat, ethanol, oxidative and alkaline pH stresses and are probably involved in cross-protection development. On the other hand, random mutagenesis of E. faecalis allowed the isolation of 10 bile salt-sensitive mutants. Their characterisation revealed that the mutation loci corresponded to genes related to DNA repair, oxidative response, transcriptional regulation, dGTP hydrolysis, membrane composition or cell wall synthesis. Further characterisation of one mutant revealed that the insertion within the E. faecalis sagA gene led to morphology changes, to perturbations of cell division and to a decrease of the resistance towards several independent physicochemical stresses.

Apolipoprotein-E genotype and the risk of developing cholelithiasis following bariatric surgery: a clue to prevention of routine prophylactic cholecystectomy.

Obesity and especially rapid weight loss following bariatric surgery are known risk factors for cholelithiasis. Since the risk may be high, prophylactic cholecystectomy has been advocated. Apolipoprotein (Apo) E, an important carrier protein in cholesterol metabolism and trafficking, is believed to play a role in gallstone pathogenesis. In particular, the Apo E4 allele has been suggested to be associated with cholesterol cholelithiasis. The aim of this study was to assess the incidence of postoperative cholelithiasis in our patient population and to determine a possible correlation with the Apo-E genotype. 134 morbidly obese patients undergoing gastric restrictive surgery [laparoscopic assisted gastric banding (LAGB) or silastic ring vertical gastroplasty (SRVG)] had abdominal ultrasound before and 6 to 12 months after operation, to determine the presence of gallstones. None of the patients enrolled in the study had gallstones before surgery. They did not have a prophylactic cholecystectomy or receive bile salt treatment. Apo-E genotypes were determined by Polymerase Chain Reaction restriction enzyme analysis. 10 patients (7.5%) developed postoperative cholelithiasis. The incidence of cholelithiasis in each ApoE genotype was: E2/E3--1/20 (5%), E3/E3--3/91 (3%), E3/E4--6/21 (29%), and E4/E4--0/2. ApoE allele frequencies in the study population were identical to those of a healthy control population. The mean BMI dropped from 43.6 to 29.4 kg/m2. The occurrence of postoperative gallstones was low in our population. However, in subjects with the Apo-E3/E4 genotype, the incidence is of practical significance. These data suggest that Apo-E genotyping may be useful in selecting patients for gallstone prevention (surgical or medical) when undergoing bariatric surgery. Further testing in larger patient populations may be able to give more definite guidelines in the future.