Qingyi decoction attenuates intestinal epithelial cell injury via the calcineurin/nuclear factor of activated T-cells pathway.

BACKGROUND: Recent studies have demonstrated that dysfunction of the intestinal barrier is a significant contributing factor to the development of severe acute pancreatitis (SAP). A stable intestinal mucosa barrier functions as a major anatomic and functional barrier, owing to the balance between intestinal epithelial cell (IEC) proliferation and apoptosis. There is some evidence that calcium overload may trigger IEC apoptosis and that calcineurin (CaN)/nuclear factor of activated T-cells (NFAT) signaling might play an important role in calcium-mediated apoptosis. AIM: To investigate the potential mechanisms underlying the therapeutic effect of Qingyi decoction (QYD) in SAP. METHODS: A rat model of SAP was created via retrograde infusion of sodium deoxycholate. Serum levels of amylase, tumor necrosis factor (TNF-α), interleukin (IL)-6, D-lactic acid, and diamine oxidase (DAO); histological changes; and apoptosis of IECs were examined in rats with or without QYD treatment. The expression of the two subunits of CaN and NFAT in intestinal tissue was measured via quantitative real-time polymerase chain reaction and western blotting. For in vitro studies, Caco-2 cells were treated with lipopolysaccharide (LPS) and QYD serum, and then cell viability and intracellular calcium levels were detected. RESULTS: Retrograde infusion of sodium deoxycholate increased the severity of pancreatic and intestinal pathology and the levels of serum amylase, TNF-α, and IL-6. Both the indicators of intestinal mucosa damage (D-lactic acid and DAO) and the levels of IEC apoptosis were elevated in the SAP group. QYD treatment reduced the serum levels of amylase, TNF-α, IL-6, D-lactic acid, and DAO and attenuated the histological findings. IEC apoptosis associated with SAP was ameliorated under QYD treatment. In addition, the protein expression levels of the two subunits of CaN were remarkably elevated in the SAP group, and the NFATc3 gene was significantly upregulated at both the transcript and protein levels in the SAP group compared with the control group. QYD significantly restrained CaN and NFATc3 gene expression in the intestine, which was upregulated in the SAP group. Furthermore, QYD serum significantly decreased the LPS-induced elevation in intracellular free Ca2+ levels and inhibited cell death. CONCLUSION: QYD can exert protective effects against intestinal mucosa damage caused by SAP and the protective effects are mediated, at least partially, by restraining IEC apoptosis via the CaN/NFATc3 pathway.

Gut microbial bile acid metabolite skews macrophage polarization and contributes to high-fat diet-induced colonic inflammation.

High-fat diet (HFD) leads to systemic low-grade inflammation, which has been involved in the pathogenesis of diverse metabolic and inflammatory diseases. Colon is thought to be the first organ suffering from inflammation under HFD conditions due to the pro-inflammatory macrophages infiltration, however, the mechanisms concerning the induction of pro-inflammatory phenotype of colonic macrophages remains unclear. In this study, we show that HFD increased the percentage of gram-positive bacteria, especially genus Clostridium, and resulted in the significant increment of fecal deoxycholic acid (DCA), a gut microbial metabolite produced by bacteria mainly restricted to genus Clostridium. Notably, reducing gram-positive bacteria with vancomycin diminished fecal DCA and profoundly alleviated pro-inflammatory macrophage infiltration in colon, whereas DCA-supplemented feedings to vancomycin-treated mice provoked obvious pro-inflammatory macrophage infiltration and colonic inflammation. Meanwhile, intra-peritoneal administration of DCA also elicited considerable recruitment of macrophages with pro-inflammatory phenotype. Mechanistically, DCA dose-dependently promoted M1 macrophage polarization and pro-inflammatory cytokines production at least partially through toll-like receptor 2 (TLR2) transactivated by M2 muscarinic acetylcholine receptor (M2-mAchR)/Src pathway. In addition, M2-mAchR mediated increase of TLR2 transcription was mainly achieved via targeting AP-1 transcription factor. Moreover, NF-κB/ERK/JNK signalings downstream of TLR2 are involved in the DCA-induced macrophage polarization. In conclusion, our findings revealed that high level DCA induced by HFD may serve as an initiator to activate macrophages and drive colonic inflammation, thus offer a mechanistic basis that modulation of gut microbiota or intervening specific bile acid receptor signaling could be potential therapeutic approaches for HFD-related inflammatory diseases.

12α-Hydroxylated bile acid induces hepatic steatosis with dysbiosis in rats.

There is an increasing need to explore the mechanism of the progression of non-alcoholic fatty liver disease. Steroid metabolism is closely linked to hepatic steatosis and steroids are excreted as bile acids (BAs). Here, we demonstrated that feeding WKAH/HkmSlc inbred rats a diet supplemented with cholic acid (CA) at 0.5 g/kg for 13 weeks induced simple steatosis without obesity. Liver triglyceride and cholesterol levels were increased accompanied by mild elevation of aminotransferase activities. There were no signs of inflammation, insulin resistance, oxidative stress, or fibrosis. CA supplementation increased levels of CA and taurocholic acid (TCA) in enterohepatic circulation and deoxycholic acid (DCA) levels in cecum with an increased ratio of 12α-hydroxylated BAs to non-12α-hydroxylated BAs. Analyses of hepatic gene expression revealed no apparent feedback control of BA and cholesterol biosynthesis. CA feeding induced dysbiosis in cecal microbiota with enrichment of DCA producers, which underlines the increased cecal DCA levels. The mechanism of steatosis was increased expression of Srebp1 (positive regulator of liver lipogenesis) through activation of the liver X receptor by increased oxysterols in the CA-fed rats, especially 4ß-hydroxycholesterol (4ßOH) formed by upregulated expression of hepatic Cyp3a2, responsible for 4ßOH formation. Multiple regression analyses identified portal TCA and cecal DCA as positive predictors for liver 4ßOH levels. The possible mechanisms linking these predictors and upregulated expression of Cyp3a2 are discussed. Overall, our observations highlight the role of 12α-hydroxylated BAs in triggering liver lipogenesis and allow us to explore the mechanisms of hepatic steatosis onset, focusing on cholesterol and BA metabolism.

Deoxycholic Acid-Mediated Sphingosine-1-Phosphate Receptor 2 Signaling Exacerbates DSS-Induced Colitis through Promoting Cathepsin B Release.

We recently have proved that excessive fecal DCA caused by high-fat diet may serve as an endogenous danger-associated molecular pattern to activate NLRP3 inflammasome and thus contributes to the development of inflammatory bowel disease (IBD). Moreover, the effect of DCA on inflammasome activation is mainly mediated through bile acid receptor sphingosine-1-phosphate receptor 2 (S1PR2); however, the intermediate process remains unclear. Here, we sought to explore the detailed molecular mechanism involved and examine the effect of S1PR2 blockage in a colitis mouse model. In this study, we found that DCA could dose dependently upregulate S1PR2 expression. Meanwhile, DCA-induced NLRP3 inflammasome activation is at least partially achieved through stimulating extracellular regulated protein kinases (ERK) signaling pathway downstream of S1PR2 followed by promoting of lysosomal cathepsin B release. DCA enema significantly aggravated DSS-induced colitis in mice and S1PR2 inhibitor as well as inflammasome inhibition by cathepsin B antagonist substantially reducing the mature IL-1ß production and alleviated colonic inflammation superimposed by DCA. Therefore, our findings suggest that S1PR2/ERK1/2/cathepsin B signaling plays a critical role in triggering inflammasome activation by DCA and S1PR2 may represent a new potential therapeutic target for the management of intestinal inflammation in individuals on a high-fat diet.

Reduction in hepatic secondary bile acids caused by short-term antibiotic-induced dysbiosis decreases mouse serum glucose and triglyceride levels.

Antibiotic-caused changes in intestinal flora (dysbiosis) can have various effects on the host. Secondary bile acids produced by intestinal bacteria are ligands for specific nuclear receptors, which regulate glucose, lipid, and drug metabolism in the liver. The present study aimed to clarify the effect of changes in secondary bile acids caused by antibiotic-induced dysbiosis on the host physiology, especially glucose, lipid, and drug metabolism. After oral administration of non-absorbable antibiotics for 5 days, decreased amounts of secondary bile acid-producing bacteria in faeces and a reduction in secondary bile acid [lithocholic acid (LCA) and deoxycholic acid (DCA)] levels in the liver were observed. Serum glucose and triglyceride levels were also decreased, and these decreases were reversed by LCA and DCA supplementation. Quantitative proteomics demonstrated that the expression levels of proteins involved in glycogen metabolism, cholesterol, bile acid biosynthesis, and drug metabolism (Cyp2b10, Cyp3a25, and Cyp51a1) were altered in the liver in dysbiosis, and these changes were reversed by LCA and DCA supplementation. These results suggested that secondary bile acid-producing bacteria contribute to the homeostasis of glucose and triglyceride levels and drug metabolism in the host, and have potential as therapeutic targets for treating metabolic disease.

High-fat Diet-induced Intestinal Hyperpermeability is Associated with Increased Bile Acids in the Large Intestine of Mice.

Metabolic syndrome is characterized by low-grade chronic systemic inflammation, which is associated with intestinal hyperpermeability. This study examined the effects of 3 high-fat diets (HFDs) composed of different fat sources (soybean oil and lard) on the intestinal permeability, tight junction (TJ) protein expression, and cecal bile acid (BA) concentrations in mice, and then analyzed their interrelations. C57/BL6 mice were fed the control diet, HFD (soybean oil), HFD (lard), and HFD (mix; containing equal concentrations of soybean oil and lard) for 8 wk. Glucose tolerance, intestinal permeability, TJ protein expression, and cecal BA concentration were evaluated. Feeding with the 3 HDFs similarly increased body weight, liver weight, and fat pad weight, and induced glucose intolerance and intestinal hyperpermeability. The expression of TJ proteins, zonula occludens-2 and junctional adhesion molecule-A, were lower in the colons of the 3 HFD groups than in the control group (P < 0.05), and these changes appeared to be related to intestinal hyperpermeability. Feeding with HFDs increased total secondary BA (SBA) and total BA concentrations along with increases in some individual BAs in the cecum. Significant positive correlations between intestinal permeability and the concentrations of most SBAs, such as deoxycholic acid and ω-muricholic acids, were detected (P < 0.05). These results suggest that the HFD-induced intestinal hyperpermeability is associated with increased BA secretion. The abundance of SBAs in the large intestine may be responsible for the hyperpermeability.

Mechanism of enhanced antiosteoporosis effect of circinal-icaritin by self-assembled nanomicelles in vivo with suet oil and sodium deoxycholate.

BACKGROUND: Circinal-icaritin (CIT), one new active aglycone of Epimedium, can exert a beneficial effect on osteoporotic bone. However, its low bioavailability limits its clinical efficacy for the treatment of osteoporosis. MATERIALS AND METHODS: In this paper, suet oil (SO) was used to improve the oral bioavailability of CIT and enhance its antiosteoporosis effect and absorption. After oral administration of CIT together with SO, the CIT and SO self-assembled into nanomicelles under the action of sodium deoxycholate (DOC) by bile secretion. The antiosteoporosis effects of the CIT-SO-DOC nanomicelles were evaluated in osteoporotic rats by bone mineral density, serum biochemical markers, bone microarchitecture, bone biomechanical properties, and related protein and gene expressions. We examined the bioavailability of CIT and its nanomicelles in vivo, and subsequently the nanomicelles were verified using transmission electron microscopy. Finally, we evaluated absorption across a rat intestinal perfusion model. RESULTS: Compared with CIT, in the CIT-SO groups, protein and messenger ribonucleic acid expressions of osteoprotegerin were increased, while expressions of receptor activator of nuclear factor-κB ligand in bone tissue were decreased; bone-turnover markers in serum of hydroxyproline, alkaline phosphatase, tartrate-resistant acid phosphatase 5b, and receptor activator of nuclear factor-κB ligand levels were decreased, while osteoprotegerin and osteocalcin levels were increased; and trabecular bone mass, microarchitecture, and bone biomechanical strength were enhanced. The relative bioavailabilities of CIT-SO high dosage, CIT-SO medium dosage, and CIT-SO low dosage (area under concentration-time curve [AUC]0-∞) compared with that of raw CIT high dosage, CIT medium dosage, and CIT low dosage (AUC0-∞) were 127%, 121%, and 134%, respectively. The average particle size of CIT-DOC was significantly decreased after adding SO (P<0.01), and the intestinal permeability coefficients of CIT-SO-DOC nanomicelles in the duodenum, jejunum, ileum, and colon were all significantly improved (P<0.01). CONCLUSION: The increased antiosteoporosis effects and bioavailability of CIT-SO-DOC self-assembled nanomicelles were due to an increase in absorption of CIT by reducing the particle sizes of CIT. SO may be a practical oral carrier for antiosteoporosis drugs with low bioavailability.

Binding of bile acids by pastry products containing bioactive substances during in vitro digestion.

The modern day consumer tends to choose products with health enhancing properties, enriched in bioactive substances. One such bioactive food component is dietary fibre, which shows a number of physiological properties including the binding of bile acids. Dietary fibre should be contained in everyday, easily accessible food products. Therefore, the aim of this study was to determine sorption capacities of primary bile acid (cholic acid - CA) and secondary bile acids (deoxycholic - DCA and lithocholic acids - LCA) by muffins (BM) and cookies (BC) with bioactive substances and control muffins (CM) and cookies (CC) in two sections of the in vitro gastrointestinal tract. Variations in gut flora were also analysed in the process of in vitro digestion of pastry products in a bioreactor. Enzymes: pepsin, pancreatin and bile salts: cholic acid, deoxycholic acid and lithocholic acid were added to the culture. Faecal bacteria, isolated from human large intestine, were added in the section of large intestine. The influence of dietary fibre content in cookies and concentration of bile acids in two stages of digestion were analysed. Generally, pastry goods with bioactive substances were characterized by a higher content of total fibre compared with the control samples. These products also differ in the profile of dietary fibre fractions. Principal Component Analysis (PCA) showed that the bile acid profile after two stages of digestion depends on the quality and quantity of fibre. The bile acid profile after digestion of BM and BC forms one cluster, and with the CM and CC forms a separate cluster. High concentration of H (hemicellulose) is positively correlated with LCA (low binding effect) and negatively correlated with CA and DCA contents. The relative content of bile acids in the second stage of digestion was in some cases above the content in the control sample, particularly LCA. This means that the bacteria introduced in the 2nd stage of digestion synthesize the LCA.

Bet v 1--a Trojan horse for small ligands boosting allergic sensitization?

BACKGROUND: Birch pollen allergy represents the main cause of winter and spring pollinosis in the temperate climate zone of the northern hemisphere and sensitization towards Bet v 1, the major birch pollen allergen, affects over 100 million allergic patients. The major birch pollen allergen Bet v 1 has been described as promiscuous acceptor for a wide variety of hydrophobic ligands. OBJECTIVE: In search of intrinsic properties of Bet v 1, which account responsible for the high allergenic potential of the protein, we thought to investigate the effects of ligand-binding on immunogenic as well as allergenic properties. METHODS: As surrogate ligand of Bet v 1 sodium deoxycholate (DOC) was selected. Recombinant and natural Bet v 1 were characterised physico-chemically as well as immunologically in the presence or absence of DOC, and an animal model of allergic sensitization was established. Moreover, human IgE binding to Bet v 1 was analysed by nuclear magnetic resonance (NMR) spectroscopy. RESULTS: Ligand-binding had an overall stabilizing effect on Bet v 1. This translated in a Th2 skewing of the immune response in a mouse model. Analyses of human IgE binding on Bet v 1 in mediator release assays revealed that ligand-bound allergen-induced degranulation at lower concentrations; however, in basophil activation tests with human basophils ligand-binding did not show this effect. For the first time, human IgE epitopes on Bet v 1 were determined using antibodies isolated from patients' sera. The IgE epitope mapping of Bet v 1 demonstrated the presence of multiple binding regions. CONCLUSIONS AND CLINICAL RELEVANCE: Deoxycholate binding stabilizes conformational IgE epitopes on Bet v 1; however, the epitopes themselves remain unaltered. Therefore, we speculate that humans are exposed to both ligand-bound and free Bet v 1 during sensitization, disclosing the ligand-binding cavity of the allergen as key structural element.

Burdock fermented by Aspergillus awamori elevates cecal Bifidobacterium, and reduces fecal deoxycholic acid and adipose tissue weight in rats fed a high-fat diet.

This study investigated the effects of dietary supplementation with burdock powder and Aspergillus awamori-fermented burdock powder at 5% on the intestinal luminal environment and body fat in rats fed a high-fat (HF) diet. Food intake and growth were unaffected by dietary manipulation. Consumption of the burdock and fermented burdock diets significantly elevated fecal IgA and mucins (indices of intestinal immune and barrier functions) and reduced fecal lithocholic acid (a risk factor for colon cancer) (p<0.05). The fermented burdock diet markedly elevated cecal Bifidobacterium and organic acids, including lactate, acetate, propionate, and butyrate, and reduced fecal deoxycholic acid (a risk factor for colon cancer) and perirenal adipose tissue weight (p<0.05), but the burdock diet did not. These results suggest that consumption of fermented burdock improves the intestinal luminal environment and suppresses obesity in rats fed a HF diet.

Enhanced phytase production from Achromobacter sp. PB-01 using wheat bran as substrate: prospective application for animal feed.

This article deals with the optimization of the various parameters for production of phytase using Achromobacter sp. PB-01 in submerged fermentation (SmF). A semisynthetic medium containing ingredients of phytase screening media (PSM) supplemented with 2% (w/v) sucrose, 1% (w/v) peptone, and 10% (w/v) wheat bran was found to be the best production medium among the various combinations tried. Among various surfactants added to SmF, Triton X-100 (0.1%) exhibited a 16% increase in phytase activity. An overall 11.2 fold enhancement in enzyme activity (0.79 U/mL→8.84 U/mL) was attained when SmF was carried out using 0.5% (v/v) inoculum of a 15 h old culture of Achromobacter sp. PB-01 at an initial pH of 5.5, temperature 30°C and allowed to grow for 48 h. Presence of accessory hydrolytic enzymes in the crude extract further added value as feed additive by mediating efficient degradation of non-starch polysaccharides (NSP). In addition, we also investigated the efficacy of phytase on different agro-industrial residues using in vitro experiments that simulated the conditions of the digestive tract. Results indicate that phytase from our source hydrolyze phytate efficiently with the concomitant liberation of inorganic phosphate, protein, reducing sugar, and calcium.

Improvement of modified charcoal-cefoperazone-deoxycholate agar by supplementation with a high concentration of polymyxin B for detection of Campylobacter jejuni and C. coli in chicken carcass rinses.

Modified charcoal-cefoperazone-deoxycholate agar (mCCDA) was improved by supplementation with a high concentration of polymyxin B. The ability of the supplemented medium to isolate Campylobacter jejuni and C. coli from chicken carcass rinses was compared to that of Campy-Cefex agar and mCCDA. Modification of mCCDA with increased polymyxin B yielded a significantly (P < 0.05) higher isolation rate and greater selectivity than those achieved using Campy-Cefex agar and mCCDA.

In vitro fermented nuts exhibit chemopreventive effects in HT29 colon cancer cells.

It is proven that nuts contain essential macro- and micronutrients, e.g. fatty acids, vitamins and dietary fibre (DF). Fermentation of DF by the gut microflora results in the formation of SCFA which are recognised for their chemopreventive potential, especially by influencing cell growth. However, little is known about cellular response to complex fermentation samples of nuts. Therefore, we prepared and analysed (pH, SCFA, bile acids, tocopherol, antioxidant capacity) fermentation supernatant (fs) fractions of nuts (almonds, macadamias, hazelnuts, pistachios, walnuts) after in vitro fermentation and determined their effects on growth of HT29 cells as well as their genotoxic/anti-genotoxic potential. The fermented nut samples contained 2- to 3-fold higher amounts of SCFA than the faeces control, but considerable reduced levels of bile acids. While most of the investigated native nuts comprised relatively high amounts of tocopherol (α-tocopherol in almonds and hazelnuts and γ- and δ-tocopherol in pistachios and walnuts), rather low concentrations were found in the fs. All nut extracts and nut fs showed a strong antioxidant potential. Furthermore, all fs, except the fs pistachio, reduced growth of HT29 cells significantly. DNA damage induced by H2O2 was significantly reduced by the fs of walnuts after 15 min co-incubation of HT29 cells. In conclusion, this is the first study which presents the chemopreventive effects (reduction of tumour-promoting desoxycholic acid, rise in chemopreventive SCFA, protection against oxidative stress) of different nuts after in vitro digestion and fermentation, and shows the potential importance of nuts in the prevention of colon cancer.

Carcinogenicity of deoxycholate, a secondary bile acid.

High dietary fat causes increased bile acid secretion into the gastrointestinal tract and is associated with colon cancer. Since the bile acid deoxycholic acid (DOC) is suggested to be important in colon cancer etiology, this study investigated whether DOC, at a high physiologic level, could be a colon carcinogen. Addition of 0.2% DOC for 8-10 months to the diet of 18 wild-type mice induced colonic tumors in 17 mice, including 10 with cancers. Addition of the antioxidant chlorogenic acid at 0.007% to the DOC-supplemented diet significantly reduced tumor formation. These results indicate that a high fat diet in humans, associated with increased risk of colon cancer, may have its carcinogenic potential mediated through the action of bile acids, and that some dietary anti-oxidants may ameliorate this carcinogenicity.

Dextran sodium sulfate enhances secretion of recombinant human transferrin in Schizosaccharomyces pombe.

The effect of medium supplementation on heterologous production of human serum transferrin (hTF) in the fission yeast Schizosaccharomyces pombe has been investigated. The productivity of recombinant hTF was low in wild-type S. pombe cells. To overcome this impediment, culture media supplements were screened for their ability to improve secretion of hTF. Casamino acids (CAA), which have been reported to increase heterologous protein productivity in Pichia pastoris, improved the secretion hTF by more than fourfold. An anion surfactant deoxycholate or polyethylene glycol also improved the secretion hTF. Interestingly, dextran sodium sulfate (DSS), a poly-anion surfactant, was found to enhance production of secreted hTF better than any other supplement tested. Addition of DSS in the presence of 2% CAA exhibited a synergistic effect on increasing hTF secretion, resulting in an increase of about sevenfold relative to conventional conditions. Cell growth was not found to be affected by the addition of DSS or CAA. DSS may act as a surfactant and may also facilitate the anchoring of liposomes, and these properties may contribute to efficient secretion or exocytosis through the plasma membrane.

Development and assessment of an innovative soil-washing process based on the use of cholic acid-derivatives as pollutant-mobilizing agents.

Surfactant-aided soil washing is often proposed for the restoration of aged organic pollutant-contaminated soils. As many of commercial surfactants have been found to be toxic and recalcitrant, the opportunity to use in this process cheap, non-toxic, and biodegradable pollutant-mobilizing agents, such as deoxycholic acid (DA), bovine bile (BB), and the residue resulting from DA extraction from BB (BBR), was studied in this work. A soil historically contaminated by chlorinated anilines and benzenes, thiophenes, and several polycyclic aromatic hydrocarbons was suspended at 15% w/v and washed in water or water amended at 1.0% (w/v) with DA, BB, BBR, or Triton X-100 (TX). The resulting effluents were supplemented with nutrients and subjected to aerobic bioremediation. The biogenic agents enhanced the water pollutant elution potential by 230/440%. TX enhanced the same parameter by about 540%; however, it mediated a lower depletion of the initial soil ecotoxicity and a more extensive mobilization of soil constituents with respect to the biogenic agents. Furthermore, TX adversely affected the biotreatability of resulting effluents, by adversely affecting the growth of cultivable bacterial biomass and the structure of eubacterial community of the effluent. On the contrary, the biogenic agents, and in particular DA and BB, enhanced the effluents bioremediation, by sustaining the growth and increasing the complexity of the effluent eubacterial communities. Thus, DA and BB are very promising additives for an effective and environmental friendly soil washing treatment of aged (chloro)organics contaminated soils.

Determination of the phospholipase activity of patatin by a continuous spectrophotometric assay.

Patatin is a family of glycoproteins that accounts for 30-40% of the total soluble protein in potato (Solanum tuberosum L.) tubers. This protein has been reported to serve as a storage protein and also to exhibit lipid phospholipase activity. This paper describes a simple continuous spectrophotometric method for assaying patatin phospholipase activity. The procedure is based on a coupled enzymatic assay using [1,2-dilinoleoyl] PC as the phospholipase substrate and lipoxygenase as the coupling enzyme. In the procedure developed in this work, lipoxygenase oxidizes the linoleic acid released by the phospholipase activity of patatin. This activity can then be followed spectrophotometrically by recording the increase in absorbance at 234 nm that results from the formation of the corresponding hydroperoxide from linoleic acid by the action of lipoxygenase. The optimal assay concentrations of patatin and lipoxygenase were established. Phospholipase activity varied with pH, reaching its optimal value at pH 9.5. Scans of the deoxycholate concentration pointed to an optimal detergent concentration of 3 mM. Phospholipid hydrolysis followed classical Michaelis-Menten kinetics (Vm = 9.8 x 10(-3) micromol/min x microg protein, Km = 7.8 microM, Vm/Km = 1.3 min(-1) x microg protein). This method proved to be specific since there was no activity in the absence of patatin. It also had the advantages of a short analysis time and the use of commercially nonradiolabeled and inexpensive substrates, which are, furthermore, natural substrates of phospholipase.

Crystal structure of a hypoallergenic isoform of the major birch pollen allergen Bet v 1 and its likely biological function as a plant steroid carrier.

Bet v 1l is a naturally occurring hypoallergenic isoform of the major birch pollen allergen Bet v 1. The Bet v 1 protein belongs to the ubiquitous family of pathogenesis-related plant proteins (PR-10), which are produced in defense-response to various pathogens. Although the allergenic properties of PR-10 proteins have been extensively studied, their biological function in plants is not known. The crystal structure of Bet v 1l in complex with deoxycholate has been determined to a resolution of 1.9A using the method of molecular replacement. The structure reveals a large hydrophobic Y-shaped cavity that spans the protein and is partly occupied by two deoxycholate molecules which are bound in tandem and only partially exposed to solvent. This finding indicates that the hydrophobic cavity may have a role in facilitating the transfer of apolar ligands. The structural similarity of deoxycholate and brassinosteroids (BRs) ubiquitous plant steroid hormones, prompted the mass spectrometry (MS) study in order to examine whether BRs can bind to Bet v 1l. The MS analysis of a mixture of Bet v 1l and BRs revealed a specific non-covalent interaction of Bet v 1l with brassinolide and 24-epicastasterone. Together, our findings are consistent with a general plant-steroid carrier function for Bet v 1 and related PR-10 proteins. The role of BRs transport in PR-10 proteins may be of crucial importance in the plant defense response to pathological situations as well as in growth and development.

Change in the positional specificity of lipoxygenase 1 due to insertion of fatty acids into phosphatidylcholine deoxycholate mixed micelles.

Linoleic and arachidonic acids were inserted into phosphatidylcholine deoxycholate mixed micelles (PDM-micelles) with their tail groups buried inside and carboxylic groups exposed outside. The fatty acid hydrophobic tail had a high affinity for the hydrophobic region of phosphatidylcholine micelles. The fatty acids inserted into phosphatidylcholine micelles were better substrates for soybean lipoxygenase 1 (LOX1) with two distinct pH optima at 7.0 and 10.0. With Tween 20-solubilized linoleic acid, the enzyme had a pH optimum at 9.0, exclusively forming 13-hydroperoxides. However, with linoleic and arachidonic acids inserted into PDM-micelles, LOX1 synthesized exclusively 9- and 5-hydroperoxides, respectively. The enzyme brought about the transformation of the substrate either at pH 7.4 or at 10.0, less efficiently at pH 10.0. However, the regioselectivity of the enzyme was not altered by increasing the pH from 7.4 to 10.0. Thus, LOX1 could utilize fatty acids bound to membranes as physiological substrates. The enzyme utilized the carboxylic group of linoleic and arachidonic acids inserted into the PDM-micelles as a recognition site to convert the compounds into 9- and 5-hydroperoxides, respectively. This was confirmed by activity measurements using methyl linoleate as the substrate. Circular dichroism measurement of LOX1 with PDM-micelles suggested that while there was a small change in the tertiary structure of LOX1, the secondary structure was unaffected. Soybean LOX1, which is arachidonate 15-LOX, acted as "5-LOX", thus making it possible to change the regiospecificity of the LOX1-catalyzed reaction by altering the physical state of the substrate.

Biodegradable lamellar particles of poly(lactide) induce sustained immune responses to a single dose of adsorbed protein.

The adjuvanticity of lamellar particles of poly(L-lactide) (PLA) towards adsorbed ovalbumin (OVA) was investigated. The aim of vaccine formulation was to maximise the amount of antigen retained on the particles and the time of retention during incubation of the formulations in PBS at 37 degrees C. Unmodified PLA lamellae were capable of adsorbing large quantities of OVA (up to 12.5% w/w) but major and rapid desorption occurred in PBS at 37 degrees C (80% released in 24 h). Retention of OVA on PLA lamellae was improved (25% released in 24 h) by precipitating the particles using aqueous sodium deoxycholate solution (DOC-modified PLA lamellae and lyophilising the lamellae-protein preparation after adsorption. Sustained immune responses were elicited in mice to a single sub-cutaneous injection of OVA adsorbed onto DOC-modified PLA lamellae. The level of antibodies induced and the pattern of response was similar to that induced by an alum-adsorbed OVA formulation. Normally boosting is required to obtain high levels of antibody when OVA is adsorbed on poly(DL-lactide co-glycolide) (PLG) microspheres. The lamellar forms of PLA may function as an efficient immunomodulator by effectively retaining adsorbed antigen and by activating immune cells due to their irregular shape. PLA lamellae have potential to stimulate enhanced immune responses to a variety of adsorbed antigens.