Breach of tolerance versus burden of bile acids: Resolving the conundrum in the immunopathogenesis and natural history of primary biliary cholangitis.

Primary biliary cholangitis (PBC) is a classic autoimmune disease due to the loss of tolerance to self-antigens. Bile acids (BA) reportedly play a major role in biliary inflammation and/or in the modulation of dysregulated immune responses in PBC. Several murine models have indicated that molecular mimicry plays a role in autoimmune cholangitis; however, they have all been limited by the relative failure to develop hepatic fibrosis. We hypothesized that species-specific differences in the BA composition between mice and humans were the primary reason for this limited pathology. Here, we aimed to study the impact of human-like hydrophobic BA composition on the development of autoimmune cholangitis and hepatic fibrosis. We took advantage of a unique construct, Cyp2c70/Cyp2a12 double knockout (DKO) mice, which have human-like BA composition, and immunized them with a well-defined mimic of the major mitochondrial autoantigen of PBC, namely 2-octynoic acid (2OA). 2OA-treated DKO mice were significantly exacerbated portal inflammation and bile duct damage with increased Th1 cytokines/chemokines at 8 weeks post-initial immunization. Most importantly, there was clear progression of hepatic fibrosis and increased expression of hepatic fibrosis-related genes. Interestingly, these mice demonstrated increased serum BA concentrations and decreased biliary BA concentrations; hepatic BA levels did not increase because of the upregulation of transporters responsible for the basolateral efflux of BA. Furthermore, cholangitis and hepatic fibrosis were more advanced at 24 weeks post-initial immunization. These results indicate that both the loss of tolerance and the effect of hydrophobic BA are essential for the progression of PBC.

Ursodeoxycholic acid impairs liver-infiltrating T-cell chemotaxis through IFN-γ and CX3CL1 production in primary biliary cholangitis.

Ursodeoxycholic acid (UDCA) is the primary treatment for primary biliary cholangitis (PBC), but its mechanism of action remains unclear. Studies suggest that UDCA enhances NF erythroid 2-related factor 2 (NFE2L2) expression and that the interaction between IFN-γ and C-X3-C motif chemokine ligand 1 (CX3CL1) facilitates biliary inflammation in PBC. Therefore, we examined the effects of UDCA on the expression of IFN-γ and CX3CL1 in in vitro and in vivo PBC models such as human liver tissue, a murine model, cell lines, and isolated human intrahepatic biliary epithelial cells (IHBECs). We observed a significant decrease in IFN-γ mRNA levels and positive correlations between IFN-γ and CX3CL1 mRNA levels post-UDCA treatment in PBC livers. NFE2L2-mediated transcriptional activation was significantly enhanced in UDCA-treated Jurkat cells. In 2-octynoic acid-immunized mice, IFN-γ production by liver-infiltrating T cells was dependent on NFE2L2 activation. IFN-γ significantly and dose-dependentlyinduced CX3CL1 expression, which was significantly decreased in HuCC-T1 cells and IHBECs upon UDCA treatment. These results suggest that UDCA-induced suppression of IFN-γ and CX3CL1 production attenuates the chemotactic and adhesive abilities of liver-infiltrating T cells in PBC.

High Expression of Fas-Associated Factor 1 Indicates a Poor Prognosis in Non-Small-Cell Lung Cancer.

Fas-associated factor 1 (FAF1) is a death-promoting protein identified as an interaction partner of the death receptor Fas. The downregulation and mutation of FAF1 have been reported in a variety of human tumors, but there have been few studies on lung cancer. Here, we investigated the prognostic significance of FAF1 expression in non-small-cell lung cancer (NSCLC), and whether aberrant FAF1 expression may be involved in the pathogenesis and prognosis of NSCLC. FAF1 expression was examined in NSCLC specimens as well as human lung cancer cell lines. In addition, changes in cell viability and apoptosis upon regulating FAF1 expression were investigated in lung cancer cell lines. As a result, high FAF1 expression was significantly associated with a poor prognosis in NSCLC. In lung cancer cell lines, FAF1 downregulation hindered cell viability and tended to promote early apoptosis. In conclusion, this is the first study of the clinical significance of FAF1 in NSCLC, showing that FAF1 overexpression is associated with a poor prognosis in NSCLC and that FAF1 acts as a dangerous factor rather than an apoptosis promoter in NSCLC.

Non-CpG sites preference in G:C > A:T transition of TP53 in gastric cancer of Eastern Europe (Poland, Romania and Hungary) compared to East Asian countries (China and Japan).

AIM: Mutation spectrum of TP53 in gastric cancer (GC) has been investigated world-widely, but a comparison of mutation spectrum among GCs from various regions in the world are still sparsely documented. In order to identify the difference of TP53 mutation spectrum in GCs in Eastern Europe and in East Asia, we sequenced TP53 in GCs from Eastern Europe, Lujiang (China), and Yokohama, Kanagawa (Japan) and identified the feature of TP53 mutations of GC in these regions. SUBJECTS AND METHOD: In total, 689 tissue samples of GC were analyzed: 288 samples from East European populations (25 from Hungary, 71 from Poland and 192 from Romania), 268 from Yokohama, Kanagawa, Japan and 133 from Lujiang, Anhui province, China. DNA was extracted from FFPE tissue of Chinese, East European cases; and from frozen tissue of Japanese GCs. PCR products were direct-sequenced by Sanger method, and in ambiguous cases, PCR product was cloned and up to 8 clones were sequenced. We used No. NC_000017.11(hg38) as the reference sequence of TP53. Mutation patterns were categorized into nine groups: six base substitutions, insertion, deletion and deletion-insertion. Within G:C > A:T mutations the mutations in CpG and non-CpG sites were divided. The Cancer Genome Atlas data (TCGA, ver.R20, July, 2019) having somatic mutation list of GCs from Whites, Asians, and other ethnicities were used as a reference for our data. RESULTS: The most frequent base substitutions were G:C > A:T transition in all the areas investigated. The G:C > A:T transition in non-CpG sites were prominent in East European GCs, compared with Asian ones. Mutation pattern from TCGA data revealed the same trend between GCs from White (TCGA category) vs Asian countries. Chinese and Japanese GCs showed higher ratio of G:C > A:T transition in CpG sites and A:T > G:C mutation was more prevalent in Asian countries. CONCLUSION: The divergence in mutation spectrum of GC in different areas in the world may reflect various pathogeneses and etiologies of GC, region to region. Diversified mutation spectrum in GC in Eastern Europe may suggest GC in Europe has different carcinogenic pathway of those from Asia.

Kinetic resolution of racemic α-tert-alkyl-α-hydroxy esters by enantiomer-selective carbamoylation.

Kinetic resolution of sterically hindered racemic α-tert-alkyl-α-hydroxy esters is performed by enantiomer-selective carbamoylation with the t-Bu-Box-Cu(II) catalyst (Box = bis(oxazoline)). The reaction with 0.5 equiv of n-C(3)H(7)NCO is carried out with a substrate-to-catalyst molar ratio of 500-5000 at -20 to 25 °C. The high enantiomer-discrimination ability of the catalyst achieves an excellent stereoselectivity factor (s = k(fast)/k(slow)) of 261 in the best case. A catalytic cycle for this reaction is proposed.

Mass spectrometric profiling of DNA adducts in the human stomach associated with damage from environmental factors.

BACKGROUND: A comprehensive understanding of DNA adducts, one of the most plausible origins of cancer mutations, is still elusive, especially in human tissues in clinical settings. Recent technological developments have facilitated the identification of multiple DNA adducts in a single experiment. Only a few attempts toward this "DNA adductome approach" in human tissues have been reported. Geospatial information on DNA adducts in human organs has been scarce. AIM: Mass spectrometry of human gastric mucosal DNA was performed to identify DNA adducts associated with environmental factors. MATERIALS AND METHODS: From 59 subjects who had received gastrectomy for gastric cancer, 306 samples of nontumor tissues and 15 samples of tumors (14 cases) were taken for DNA adductome analysis. Gastric nontumor tissue from autopsies of 7 subjects without gastric cancer (urothelial cancer, hepatocellular carcinoma, lung cancer each; the other four cases were without any cancers) was also investigated. Briefly, DNA was extracted from each sample with antioxidants, digested into nucleosides, separated by liquid chromatography, and then electrospray-ionized. Specific DNA adducts were identified by mass/charge number and column retention time compared to standards. Information on lifestyle factors such as tobacco smoking and alcohol drinking was taken from the clinical records of each subject. RESULTS: Seven DNA adducts, including modified bases, C5-methyl-2'-deoxycytidine, 2'-deoxyinosine, C5-hydroxymethyl-2'-deoxycytidine, N6-methyl-2'-deoxyadenosine, 1,N6-etheno-2'-deoxyadenosine, N6-hydroxymethyl-2'-deoxyadenosine, and C8-oxo-2'-deoxyguanosine, were identified in the human stomach and characterized. Intraindividual differences according to the multiple sites of these adducts were noted but were less substantial than interindividual differences. N6-hydroxymethyl-2'-deoxyadenosine was identified in the human stomach for the first time. The amount of C5-hydroxymethyl-2'-deoxycytidine was higher in the stomachs of subjects without gastric cancer than in the nontumor and tumor portions of the stomach in gastric cancer patients. Higher levels of 1,N6-etheno-2'-deoxyadenosine were detected in the subjects who reported both smoking and drinking than in those without these habits. These DNA adducts showed considerable correlations with each other. CONCLUSIONS: We characterized 7 DNA adducts in the nontumor portion of the human stomach in both gastric cancer subjects and nongastric cancer subjects. A reduction in C5-hydroxymethyl-dC even in the nontumor mucosa of patients with gastric cancer was observed. Smoking and drinking habits significantly influenced the quantity of one of the lipid peroxidation-derived adducts, etheno-dA. A more expansive DNA adductome profile would provide a comprehensive understanding of the origin of human cancer in the future.

Geospatial Assessments of DNA Adducts in the Human Stomach: A Model of Field Cancerization.

BACKGROUND: Field cancerization is a popular concept regarding where cancer cells arise in a plane, such as the opened-up gastrointestinal mucosa. The geospatial distribution of DNA adducts, some of which are believed to initiate mutation, may be a clue to understanding the landscape of the preferred occurrence of gastric cancer in the human stomach, such that the occurrence is much more frequent in the lesser curvature than in the greater curvature. METHODS: Seven DNA adducts, C5-methyl-2'-deoxycytidine, 2'-deoxyinosine, C5-hydroxymethyl-2'-deoxycytidine, N6-methyl-2'-deoxyadenosine, 1,N6-etheno-2'-deoxyadenosine, N6-hydroxymethyl-2'-deoxyadenosine, and C8-oxo-2'-deoxyguanosine, from different points and zones of the human stomach were semi quantitatively measured by liquid chromatography/tandem mass spectrometry. The differences in the quantity of these DNA adducts from the lesser and greater curvature, the upper, middle and lower third zones, the anterior and posterior wall of the stomach, and the mucosae distant from and near the tumor were compared to determine whether the location preference of cancer in the stomach could be explained by the distribution of these DNA adducts. Comparisons were conducted considering the tumor locations and operation methods. CONCLUSIONS: Regarding the DNA adducts investigated, significant differences in quantities and locations in the whole stomach were not noted; thus, these DNA adducts do not explain the preferential occurrence of cancer in particular locations of the human stomach.

Synthesis of isoindolobenzazepine alkaloids based on radical reactions or Pd(0)-catalyzed reactions.

Methods for synthesis of a ring system characteristic of isoindolobenzazepine alkaloids were studied. Synthesis of lennoxamine and a formal synthesis of chelenine were accomplished in a short route via radical or Pd(0)-catalyzed cyclization as the key step. An altenative approach based on a radical migration of a cyano group or Pd(0)-catalyzed carbonylation was also developed for both alkaloids.

Asymmetric hydrogenation of aromatic ketones catalyzed by the TolBINAP/DMAPEN-ruthenium(II) complex: a significant effect of N-substituents of chiral 1,2-diamine ligands on enantioselectivity.

Asymmetric hydrogenation of acetophenone in the presence of Ru(II) catalysts coordinated by TolBINAP and a series of chiral 1,2-diamines was studied. The sense and degree of enantioselectivity were highly dependent on the N-substituents of the diamine ligands. The N-substituent effect was discussed in detail. Among these catalysts, the (S)-TolBINAP/(R)-DMAPEN-Ru(II) complex showed the highest enantioselectivity. The mode of enantioface selection was interpreted by using transition state models based on the X-ray structure of the catalyst precursor. The chiral catalyst effected the hydrogenation of alkyl aryl ketones and arylglyoxal dialkyl acetals to afford the chiral alcohol in >99% ee in the best cases. Hydrogenation of racemic benzoin methyl ether with the chiral catalyst through dynamic kinetic resolution gave the anti-alcohol (syn:anti = 3:97) in 98% ee, while the reaction of alpha-amidopropiophenones resulted in the syn-alcohols (syn:anti = 96:4 to >99:1) in >98% ee.

General asymmetric hydrogenation of alpha-branched aromatic ketones catalyzed by TolBINAP/DMAPEN-ruthenium(II) complex.

[reaction: see text] A catalyst system consisting of RuCl2[(S)-tolbinap][(R)-dmapen] and t-C4H9OK in 2-propanol effects asymmetric hydrogenation of arylglyoxal dialkylacetals to give the alpha-hydroxy acetals in up to 98% ee. Hydrogenation of racemic alpha-amidopropiophenones under dynamic kinetic resolution predominantly gives the syn alcohols in up to 99% ee and >98% de, while the reaction of racemic bezoin methyl ether gives the anti alcohols in excellent stereoselectivity.

Asymmetric conjugate hydrocyanation of α,ß-unsaturated N-acylpyrroles with the Ru(phgly)2(binap)-CH3OLi catalyst system.

Asymmetric conjugate hydrocyanation of α,ß-unsaturated carboxylic acid derivatives catalyzed by a Ru[(S)-phgly]2[(S)-binap]-CH3OLi system was examined. The N-acylpyrrole gave the best result in terms of reactivity and enantioselectivity. A series of substrates with alkyl or heterosubstituted alkyl groups at the ß-position reacted with a substrate-to-catalyst molar ratio of 200-2000 to afford the ß-cyano products in the range of 88%->99% ee. The mode of enantioselection in the hydrocyanation was proposed.

Enantioselective hydrocyanation of N-protected aldimines.

Enantioselective hydrocyanation of N-benzyloxycarbonyl aldimines catalyzed by a Ru[(S)-phgly](2)[(S)-binap]/C(6)H(5)OLi system or a bimetallic complex [Li{Ru[(S)-phgly](2)[(S)-binap]}]Cl affords the amino nitriles in 92-99% ee. The reaction is carried out in tert-C(4)H(9)OCH(3) with a substrate-to-catalyst molar ratio in the range of 500-5000 at -20 to 0 °C. Primary, secondary, and tertiary alkyl imines as well as the aryl and heteroaryl substrates are smoothly cyanated to produce the desired products in high yield.

Enantioselective hydrocyanation of aldehydes catalyzed by [Li{Ru(phgly)2(binap)}]X (X = Cl, Br).

Novel bimetallic complexes [Li{Ru[(S)-phgly](2)[(S)-binap]}]X (X = Cl, Br) are readily synthesized by mixing Ru[(S)-phgly](2)[(S)-binap] and LiX. A single-crystal X-ray analysis reveals the structure. These bimetallic complexes efficiently catalyze asymmetric hydrocyanation of aldehydes with a substrate-to-catalyst molar ratio of 500-2000 at -78 to -60 °C. A range of aromatic, heteroaromatic, and α,ß-unsaturated aldehydes as well as a tert-alkyl aldehyde is converted to the cyanohydrins in high enantiomeric excess (up to 99%).

Enantiomer-selective carbamoylation of racemic alpha-hydroxy gamma-lactones with chiral Cu(II) Catalysts: an example of a highly active Lewis acid catalyzed reaction.

Enantiomer-selective carbamoylation of racemic alpha-hydroxy gamma-lactones with half equivalents of isocyanates in the presence of chiral Cu(II) catalysts was studied. Among a series of catalyst bearing chiral bis(oxazoline) (box) and pyridine(bisoxazoline) ligands, [Cu(tBu-box)]X(2) [X=OSO(2)CF(3) (3 a), SbF(6) (3 b)] showed the highest enantioselectivity in the reaction of pantolactone (1 a). Use of n-C(3)H(7)NCO, a small alkyl isocyanate, in CH(2)Cl(2) solution was important to achieve a high level of enantiomer selection. The tBu-box-Cu(II) catalyst efficiently differentiated two enantiomers of beta-substituted alpha-hydroxy gamma-lactones under the optimized reaction conditions, resulting in a stereoselectivity factor (s=k(fast)/k(slow)) of up to 209. Furthermore, this catalyst is highly active, so that the carbamoylation can be conducted with a substrate-to-catalyst molar ratio of 2000-3000. A catalytic cycle of this reaction is also proposed.

On the synthesis of protopine alkaloids.

For the synthesis of protopine alkaloids, we studied a reaction sequence based on a ring enlargement of indeno[2,1-a][3]benzazepines by a singlet oxygen oxygenation, followed by conversion of an amide carbonyl group of the resultant 10-membered keto-lactam to a methylene group, which is the last step for completion of the synthesis. The key substances, indeno[2,1-a][3]benzazepines, were prepared by Bischler-Napieralski cyclization of alkoxy-substituted 1-(2-bromobenzyl)-3-benzazepin-2-ones. Steric effects of the substituents in this synthesis were examined.

Lithium chloride: an active and simple catalyst for cyanosilylation of aldehydes and ketones.

LiCl acts as a highly effective catalyst for cyanosilylation of various aldehydes and ketones to the corresponding silylated cyanohydrins. The reaction proceeds smoothly with a substrate/catalyst molar ratio of 100-100,000 at 20-25 degrees C under solvent-free conditions. alpha,beta-Unsaturated aldehydes are completely converted to the 1,2-adducts. The cyanation products can be isolated by direct distillation of the reaction mixture.