Anti-Atopic Dermatitis Effects of Abietic Acid Isolated from Rosin under Condition Optimized by Response Surface Methodology in DNCB-Spread BALB/c Mice.

Abietic acid (AA) is known to have beneficial effects on inflammation, photoaging, osteoporosis, cancer, and obesity; however, its efficacy on atopic dermatitis (AD) has not been reported. We investigated the anti-AD effects of AA, which was newly isolated from rosin, in an AD model. To achieve this, AA was isolated from rosin under conditions optimized by response surface methodology (RSM), and its effects on cell death, iNOS-induced COX-2 mediated pathway, inflammatory cytokine transcription, and the histopathological skin structure were analyzed in 2,4-dinitrochlorobenzene (DNCB)-treated BALB/c mice after treatment with AA for 4 weeks. AA was isolated and purified through isomerization and reaction-crystallization under the condition (HCl, 2.49 mL; reflux extraction time, 61.7 min; ethanolamine, 7.35 mL) established by RSM, resulting in AA with a purity and extraction yield of 99.33% and 58.61%, respectively. AA exhibited high scavenging activity against DPPH, ABTS, and NO radicals as well as hyaluronidase activity in a dose-dependent manner. The anti-inflammatory effects of AA were verified in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages through amelioration of the inflammatory response, including NO production, iNOS-induced COX-2 mediated pathway activation, and cytokine transcription. In the DNCB-treated AD model, the skin phenotypes, dermatitis score, immune organ weight, and IgE concentration were significantly ameliorated in the AA cream (AAC)-spread groups compared to the vehicle-spread group. In addition, AAC spread ameliorated DNCB-induced deterioration of skin histopathological structure through the recovery of the thickness of the dermis and epidermis and the number of mast cells. Furthermore, activation of the iNOS-induced COX-2 mediated pathway and increased inflammatory cytokine transcription were ameliorated in the skin of the DNCB+AAC-treated group. Taken together, these results indicate that AA, newly isolated from rosin, exhibits anti-AD effects in DNCB-treated AD models, and has the potential to be developed as a treatment option for AD-related diseases.

Therapeutic effect of ethyl acetate extract from Asparagus cochinchinensis on phthalic anhydride-induced skin inflammation.

Asparagus cochinchinensis has been used to treat various diseases including fever, cough, kidney disease, breast cancer, inflammatory disease and brain disease, while IL-4 cytokine has been considered as key regulator on the skin homeostasis and the predisposition toward allergic skin inflammation. However, few studies have investigated its effects and IL-4 correlation on skin inflammation to date. To quantitatively evaluate the suppressive effects of ethyl acetate extracts of A. cochinchinensis (EaEAC) on phthalic anhydride (PA)-induced skin inflammation and investigate the role of IL-4 during their action mechanism, alterations in general phenotype biomarkers and luciferase-derived signals were measured in IL-4/Luc/CNS-1 transgenic (Tg) mice with PA-induced skin inflammation after treatment with EaEAC for 2 weeks. Key phenotype markers including lymph node weight, immunoglobulin E (IgE) concentration, epidermis thickness and number of infiltrated mast cells were significantly decreased in the PA+EaEAC treated group compared with the PA+Vehicle treated group. In addition, expression of IL-1ß and TNF-α was also decreased in the PA+EaEAC cotreated group, compared to PA+Vehicle treated group. Furthermore, a significant decrease in the luciferase signal derived from IL-4 promoter was detected in the abdominal region, submandibular lymph node and mesenteric lymph node of the PA+EaEAC treated group, compared to PA+Vehicle treated group. Taken together, these results suggest that EaEAC treatment could successfully improve PA-induced skin inflammation of IL-4/Luc/CNS-1 Tg mice, and that IL-4 cytokine plays a key role in the therapeutic process of EaEAC.

Helicobacter pylori cholesteryl α-glucosides contribute to its pathogenicity and immune response by natural killer T cells.

Approximately 10-15% of individuals infected with Helicobacter pylori will develop ulcer disease (gastric or duodenal ulcer), while most people infected with H. pylori will be asymptomatic. The majority of infected individuals remain asymptomatic partly due to the inhibition of synthesis of cholesteryl α-glucosides in H. pylori cell wall by α1,4-GlcNAc-capped mucin O-glycans, which are expressed in the deeper portion of gastric mucosa. However, it has not been determined how cholesteryl α-glucosyltransferase (αCgT), which forms cholesteryl α-glucosides, functions in the pathogenesis of H. pylori infection. Here, we show that the activity of αCgT from H. pylori clinical isolates is highly correlated with the degree of gastric atrophy. We investigated the role of cholesteryl α-glucosides in various aspects of the immune response. Phagocytosis and activation of dendritic cells were observed at similar degrees in the presence of wild-type H. pylori or variants harboring mutant forms of αCgT showing a range of enzymatic activity. However, cholesteryl α-glucosides were recognized by invariant natural killer T (iNKT) cells, eliciting an immune response in vitro and in vivo. Following inoculation of H. pylori harboring highly active αCgT into iNKT cell-deficient (Jα18(-/-)) or wild-type mice, bacterial recovery significantly increased in Jα18(-/-) compared to wild-type mice. Moreover, cytokine production characteristic of Th1 and Th2 cells dramatically decreased in Jα18(-/-) compared to wild-type mice. These findings demonstrate that cholesteryl α-glucosides play critical roles in H. pylori-mediated gastric inflammation and precancerous atrophic gastritis.

Bioconversion of piceid to piceid glucoside using amylosucrase from Alteromonas macleodii deep ecotype.

Resveratrol, or its glycoside form piceid, is a dietary antioxidant polyphenolic compound, found in grapes and red wine that has been shown to have protective effects against cardiovascular disease. However, very low water solubility of the compound may limit its application in the food and pharmaceutical industries. The amylosucrase (AMAS) of Alteromonas macleodii Deep ecotype was expressed in Escherichia coli and showed high glycosyltransferase activity to produce the glucosyl piceid when piceid was used as an acceptor. The conversion yield of piceid glucoside was 35.2%. Biotransformation using culture of the E. coli harboring the amas gene increased the yield up to 70.8%. The transfer product was purified by reverse phase chromatography and recycling preparative HPLC, and the molecular structure of the piceid glucoside was determined using NMR spectroscopy. The piceid glucoside was identified as glucosyl-alpha-(1-->4)-piceid. The solubility of glucosyl piceid was 5.26 and 1.14 times higher than those of resveratrol and piceid, respectively. It is anticipated that dietary intake of this compound is more effective by enhancing the bioavailability of resveratrol in the human body because of its hydrophilic properties in the intestinal fluid.

Membrane-associated activation of cholesterol α-glucosyltransferase, an enzyme responsible for biosynthesis of cholesteryl-α-D-glucopyranoside in Helicobacter pylori critical for its survival.

Helicobacter pylori (H. pylori) is the causative pathogen underlying gastric diseases such as chronic gastritis and gastric cancer. Previously, the authors revealed that α1,4-linked N-acetylglucosamine-capped O-glycan (αGlcNAc) found in gland mucin suppresses H. pylori growth and motility by inhibiting catalytic activity of cholesterol α-glucosyltransferase (CHLαGcT), the enzyme responsible for biosynthesis of the major cell wall component cholesteryl-α-D-glucopyranoside (CGL). Here, the authors developed a polyclonal antibody specific for CHLαGcT and then undertook quantitative ultrastructural analysis of the enzyme's localization in H. pylori. They show that 66.3% of CHLαGcT is detected in the cytoplasm beneath the H. pylori inner membrane, whereas 24.7% is present on the inner membrane. In addition, 2.6%, 5.0%, and 1.4% of the protein were detected in the periplasm, on the outer membrane, and outside microbes, respectively. By using an in vitro CHLαGcT assay with fractionated H. pylori proteins, which were used as an enzyme source for CHLαGcT, the authors demonstrated that the membrane fraction formed CGL, whereas other fractions did not. These data combined together indicate that CHLαGcT is originally synthesized in the cytoplasm of H. pylori as an inactive form and then activated when it is associated with the cell membrane. This article contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Crystal structure and functional insight of HP0420-homolog from Helicobacter felis.

Helicobacter pylori infect more than half of the world's population and are considered a cause of peptic ulcer disease and gastric cancer. Recently, hypothetical gene HP0421 was identified in H. pylori as a cholesterol alpha-glucosyltransferase, which is required to synthesize cholesteryl glucosides, essential cell wall components of the bacteria. In the same gene-cluster, HP0420 was co-identified, whose function remains unknown. Here we report the crystal structure of HP0420-homolog of H. felis (HF0420) to gain insight into the function of HP0420. The crystal structure, combined with size-exclusion chromatography, reveals that HF0420 adopts a homodimeric hot-dog fold. The crystal structure suggests that HF0420 has enzymatic activity that involves a conserved histidine residue at the end of the central alpha-helix. Subsequent biochemical studies provide clues to the function of HP0420 and HF0420.

Carbohydrate-dependent defense mechanisms against Helicobacter pylori infection.

Helicobacter pylori is a Gram-negative bacterium that infects over 50% of the world's population. This organism causes various gastric diseases such as chronic gastritis, peptic ulcer, and gastric cancer. H. pylori possesses lipopolysaccharide, which shares structural similarity to Lewis blood group antigens in gastric mucosa. Such antigenic mimicry could result in immune tolerance against antigens of this pathogen. On the other hand, H. pylori colonize gastric mucosa by utilizing adhesins, which bind Lewis blood group antigen-related carbohydrates expressed on gastric epithelial cells. In chronic gastritis, lymphocytes infiltrate the lamina propria, and such infiltration is facilitated by 6-sulfo sialyl Lewis X-capped O-glycans, peripheral lymph node addressin (PNAd), on high endothelial venule (HEV)-like vessels. The number of HEV-like vessels increases as chronic inflammation progresses. Furthermore, PNAd formed on HEV-like vessels disappear once H. pylori is eradicated. These results indicate that PNAd plays an important role in H. pylori-associated inflammation. H. pylori barely colonizes gland mucous cell-derived mucin where alpha1,4-GlcNAc-capped O-glycans exist. In vitro experiments show that alpha1,4-GlcNAc-capped O-glycans function as a natural antibiotic to inhibit H. pylori growth. We recently identified cholesterol alpha-glucosyltransferase (CHLalphaGcT) using an expression cloning strategy and showed that this enzyme is specifically inhibited by mucin-type O-glycans like those present in deeper portions of the gastric mucosa. These findings show that a battery of carbohydrates expressed in the stomach is closely associated with pathogenesis and also prevention of H. pylori-related diseases.

Roles of gastric mucin-type O-glycans in the pathogenesis of Helicobacter pylori infection.

Helicobacter pylori is a Gram-negative bacterium that infects over 50% of the world's population. This organism causes various gastric diseases such as chronic gastritis, peptic ulcer, and gastric cancer. H. pylori possesses lipopolysaccharides that share structural similarity to Lewis blood group antigens in gastric mucosa. Such antigenic mimicry could result in immune tolerance against antigens of this pathogen. On the other hand, H. pylori colonizes gastric mucosa by utilizing adhesins that bind Lewis blood group antigen-related carbohydrates expressed on gastric epithelial cells. After colonization, H. pylori induces acute inflammatory responses mainly by neutrophils. This acute phase is gradually replaced by a chronic inflammatory response. In chronic gastritis, lymphocytes infiltrate the lamina propria, and such infiltration is facilitated by the interaction between L-selectin on lymphocytes and peripheral lymph node addressin (PNAd), which contains 6-sulfo sialyl Lewis X-capped O-glycans, on high endothelial venule (HEV)-like vessels. H. pylori barely colonizes gland mucous cell-derived mucin where alpha1,4-GlcNAc-capped O-glycans exist. In vitro experiments show that alpha1,4-GlcNAc-capped O-glycans function as a natural antibiotic to inhibit H. pylori growth. These findings show that distinct sets of carbohydrates expressed in the stomach are closely associated with pathogenesis and prevention of H. pylori-related diseases, providing therapeutic potentialities based on specific carbohydrate modulation.

Alpha1,4GlcNAc-capped mucin-type O-glycan inhibits cholesterol alpha-glucosyltransferase from Helicobacter pylori and suppresses H. pylori growth.

Helicobacter pylori infects over half of the world's population and is thought to be a leading cause of gastric ulcer, gastric carcinoma, and gastric malignant lymphoma of mucosa-associated lymphoid tissue type. Previously, we reported that a gland mucin (MUC6) present in the lower portion of the gastric mucosa containing alpha1,4-N-acetylglucosamine (alpha1,4GlcNAc)-capped core 2-branched O-glycans suppresses H. pylori growth by inhibiting the synthesis of alpha-glucosyl cholesterol, a major constituent of the H. pylori cell wall (Kawakubo et al. 2004. Science. 305:1003-1006). Therefore, we cloned the genomic DNA encoding cholesterol alpha-glucosyltransferase (HP0421) and expressed its soluble form in Escherichia coli. Using this soluble HP0421, we show herein that HP0421 sequentially acts on uridine diphosphoglucose and cholesterol in an ordered Bi-Bi manner. We found that competitive inhibition of HP0421 by alpha1,4GlcNAc-capped core 2-branched O-glycan is much more efficient than noncompetitive inhibition by newly synthesized alpha-glucosyl cholesterol. Utilizing synthetic oligosaccharides, alpha-glucosyl cholesterol, and monosaccharides, we found that alpha1,4GlcNAc-capped core 2-branched O-glycan most efficiently inhibits H. pylori growth. These findings together indicate that alpha1,4GlcNAc-capped O-glycans suppress H. pylori growth by inhibiting HP0421, and that alpha1,4GlcNAc-capped core 2 O-glycans may be useful to treat patients infected with H. pylori.

A distinctive set of genes is upregulated during the inflammation-carcinoma sequence in mouse stomach infected by Helicobacter felis.

Helicobacter pylori infects over half the population worldwide and is a leading cause of chronic gastritis and gastric cancer. However, the mechanism by which this organism induces inflammation and carcinogenesis is not fully understood. In the present study we used insulin-gastrin (INS-GAS) transgenic mice that fully develop gastric adenocarcinoma after infection of H. pylori-related Helicobacter felis. Histological examination revealed that more than half of those mice developed invasive adenocarcinoma after 8 months of infection. These carcinomas were stained by NCC-ST-439 and HECA-452 that recognize 6-sulfated and non-sulfated sialyl Lewis X. Lymphocytic infiltration predominantly to submucosa was observed in most H. felis-infected mice, and this was associated with the formation of peripheral lymph node addressin (PNAd) on high endothelial venule (HEV)-like vessels detected by MECA-79. Time-course analysis of gene expression by using gene microarray revealed upregulation of several inflammation-associated genes including chemokines, adhesion molecules, surfactant protein D (SP-D), and CD74 in the infected stomach. Immunohistochemical analysis demonstrated that SP-D is expressed in hyperplasia and adenocarcinoma whereas CD74 is expressed in adenocarcinoma in situ and invasive carcinoma. These results as a whole indicate that H. felis induces HEV-like vessels and inflammation-associated chemokines and chemokine receptors, followed by adenocarcinoma formation.

Assay of human gastric mucin as a natural antibiotic against Helicobacter pylori.

Helicobacter pylori infects more than half of the world's population and is considered a leading cause of peptic ulcer and gastric carcinoma. Although a large number of persons are infected with H. pylori, only a limited number of those infected (approximately 3%) develop peptic ulcers and gastric carcinoma. The progression of the disease is restricted by deeper portion of the gastric mucosa, and in many persons glandular atrophy appears to be prevented by mucins secreted in the deeper portion of the mucosa. Recent studies have shown that this inhibitory activity is at least partly due to the expression of alpha1,4-N-acetylglucosamine residues attached to the mucin (MUC6) in the deeper portion of the mucosa. alpha1,4-N-acetylglucosamine residues inhibit cholesterol alpha-glucosyltransferase, the product of which constitutes a major component of H. pylori cell wall. This inhibitory activity is thus regarded as a natural antibiotic function. This chapter describes the assay for antibiotic activity of MUC6 mucin against H. pylori infection and growth as well as inhibition by alpha1,4-N-acetylglucosamine-capped mucin-type oligosaccharides.

Expression cloning of cholesterol alpha-glucosyltransferase, a unique enzyme that can be inhibited by natural antibiotic gastric mucin O-glycans, from Helicobacter pylori.

Helicobacter pylori infects over half the world's population, but only 3% of those infected develop peptic ulcer, gastric cancer, and mucosa-associated lymphoid tissue (MALT) lymphoma. In H. pylori, alpha-glucosyl cholesterol constitutes more than 25% of cell wall lipids, and it has been suggested that alpha-glucosyl cholesterol is essential for H. pylori viability. Here, we identified cholesterol alpha-glucosyltransferase (CHLalphaGcT) using an expression cloning strategy and showed that this enzyme is distinctively inhibited by mucin-type O-glycans similar to those present in deeper portions of the gastric mucosa. Moreover, inactivation of CHLalphaGcT by homologous recombination led to H. pylori lethality. These results indicate that H. pylori CHLalphaGcT is a unique enzyme targeted by a natural antibiotic mucin and constitutes an excellent therapeutic target to prevent H. pylori-induced peptic ulcer, gastric carcinoma, and MALT lymphoma.