Extensive gene flow over Europe and possible speciation over Eurasia in the ectomycorrhizal basidiomycete Laccaria amethystina complex

Biogeographical patterns and large-scale genetic structure have been little studied in ectomycorrhizal (EM) fungi, despite the ecological and economic importance of EM symbioses. We coupled population genetics and phylogenetic approaches to understand spatial structure in fungal populations on a continental scale. Using nine microsatellite markers, we characterized gene flow among 16 populations of the widespread EM basidiomycete Laccaria amethystina over Europe (i.e. over 2900 km). We also widened our scope to two additional populations from Japan (10(4) km away) and compared them with European populations through microsatellite markers and multilocus phylogenies, using three nuclear genes (NAR, G6PD and ribosomal DNA) and two mitochondrial ribosomal genes. European L. amethystina populations displayed limited differentiation (average F(ST) = 0.041) and very weak isolation by distance (IBD). This panmictic European pattern may result from effective aerial dispersal of spores, high genetic diversity in populations and mutualistic interactions with multiple hosts that all facilitate migration. The multilocus phylogeny based on nuclear genes confirmed that Japanese and European specimens were closely related but clustered on a geographical basis. By using microsatellite markers, we found that Japanese populations were strongly differentiated from the European populations (F(ST) = 0.416), more than expected by extrapolating the European pattern of IBD. Population structure analyses clearly separated the populations into two clusters, i.e. European and Japanese clusters. We discuss the possibility of IBD in a continuous population (considering some evidence for a ring species over the Northern Hemisphere) vs. an allopatric speciation over Eurasia, making L. amethystina a promising model of intercontinental species for future studies.     

Loss of density-dependent growth inhibition and dissociation of α-catenin from E-cadherin

Normal human breast epithelial (HBE) cells at early (9th) passage ceased growth and formed a monolayer when they reached confluence. Immunostaining and Western blotting revealed that α- and β-catenins colocalized and coprecipitated with E-cadherin, suggesting a complex formation of E-cadherin with α- and β-catenins in early passage cells. In contrast, HBE cells at late (12–13th) passage did not cease growth after confluence but stratified. The late passage cells exhibited enhanced colony forming ability in soft agar compared with early passage cells, however, they had a definite proliferating lifespan and were primarily diploid. In late passage cells grown as multilayers, α-catenin was expressed but did not colocalize or coprecipitate with E-cadherin, suggesting its dissociation from E-cadherin. Coimmunoprecipitation of α-actinin with α-catenin suggested an indirect link between the E-cadherin-β-catenin complex and α-actinin via α-catenin in early, but not in late passage cells. β-Catenin in late passage cells was tyrosine phosphorylated and was not dephosphorylated following the addition of inhibitors of tyrosine kinases. Inhibition of dephosphorylation of β-catenin in early passage cells by vanadate, an inhibitor of protein tyrosine phosphatases, caused overgrowth of cells beyond the saturation density and loss of α-catenin from the E-cadherin-β-catenin complex. The results suggest that E-cadherin requires its association with α-actinin-associated α-catenin to maintain epithelial monolayers and accomplish the density-dependent inhibition of growth. In addition, association between E-cadherin and α-catenin is suggested to be prevented by the presence of tyrosine phosphorylated β-catenin which associates with E-cadherin. J. Cell. Physiol. 173:54–63, 1997. © 1997 Wiley-Liss, Inc.     

4-Amino-2-cyanopyrimidines: novel scaffold for nonpeptidic cathepsin S inhibitors

We describe here a novel 4-amino-2-cyanopyrimidine scaffold for nonpeptidomimetic cathepsin S selective inhibitors. Some of the synthesized compounds have sub-nanomolar potency and high selectivity toward cathepsin S along with promising pharmacokinetic and physicochemical properties. The key structural features of the inhibitors consist of a combination of a spiro[2.5]oct-6-ylmethylamine P2 group at the 4-position, a small or polar P3 group at the 5-position and/or a polar group at the 6-position of the pyrimidine.     

Enzymatic synthesis of oligosaccharide containing Le(x) unit by using partially purified chicken serum

Animal sera were screened for an alternative enzyme source of alpha1,3-fucosyltransferase, and the highest activity was observed in chicken serum. A partially purified enzyme fraction almost devoid of coexisting glycosidases was prepared from the chicken serum, and used for the fucosylation of LacNAc compounds. The enzyme reaction was efficient enough to allow the one-pot preparation of designed Le(x) compounds such as LNFP III.     

Bacillus subtilis ypgA gene is fni, a nonessential gene encoding type 2 isopentenyl diphosphate isomerase

We previously identified the fni gene of Streptomyces sp. strain CL190 as type 2 isopentenyl diphosphate (IPP) isomerase, which needs both FMN and NADPH for enzyme activity. An fni gene homolog, ypgA, was detected in the database of the Bacillus subtilis genome. However, the ypgA product was about 140 amino acids shorter in the N-terminal than the Streptomyces fni gene product. A database search found three new putative start codons in 129, 225, and 411 bases upstream of the original start codon of the ypgA gene. The longest gene product, which was named ypgA3, showed the most significant homology to the Streptomyces fni gene product. The ypgA3 gene was expressed with an N-terminal His-tag in Escherichia coli and the purified soluble protein was characterized in detail. The ypgA3 protein converted IPP to its isomer dimethylallyl diphosphate in the presence of both FMN and NADPH. The enzyme also catalyzed the reverse reaction in the presence of both the cofactors. Disruption of the ypgA3 gene was not lethal to B. subtilis. These results indicate that Bacillus ypgA3 gene is fni, a nonessential gene encoding type 2 IPP isomerase.     

5Alpha-bile alcohols function as farnesoid X receptor antagonists

The farnesoid X receptor (FXR) is a bile acid/alcohol-activated nuclear receptor that regulates lipid homeostasis. Unlike other steroid receptors, FXR binds bile acids in an orientation that allows the steroid nucleus A ring to face helix 12 in the receptor, a crucial domain for coactivator-recruitment. Because most naturally occurring bile acids and alcohols contain a cis-oriented A ring, which is distinct from that of other steroids and cholesterol metabolites, we investigated the role of this 5beta-configuration in FXR activation. The results showed that the 5beta-(A/B cis) bile alcohols 5beta-cyprinol and bufol are potent FXR agonists, whereas their 5alpha-(A/B trans) counterparts antagonize FXR transactivation and target gene expression. Both isomers bound to FXR, but their ability to induce coactivator-recruitment and thereby induce transactivation differed. These findings suggest a critical role for the A-ring orientation of bile salts in agonist/antagonist function.     

Highly sensitive detection of E2 activity in ubiquitination using an artificial RING finger

The ubiquitin‐conjugating (E2) enzymes of protein ubiquitination are associated with various diseases such as leukemia, lung cancer, and breast cancer. Rapid and accurate detection of E2 enzymatic activities remains poor. Here, we described the detection of E2 activity on a signal accumulation ISFET biosensor (AMIS sensor) using an artificial RING finger (ARF). The use of ARF enables the simplified detection of E2 activity without a substrate. The high‐sensitivity quantitative detection of E2 activities was demonstrated via real‐time monitoring over a response range of femtomolar to micromolar concentrations. Furthermore, the monitoring of E2 activities was successfully achieved using human acute promyelocytic leukemia cells following treatment with the anticancer drug bortezomib, which allowed the assessment of the pathological conditions. This strategy is extremely simple and convenient, and the present detection could be widely applied to specific E2s for various types of cancers. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

L-MBP is expressed in epithelial cells of mouse small intestine

The mannan-binding proteins (L-MBP and S-MBP, also denoted MBL-C and MBL-A), mainly produced in liver and existing in liver and serum, play important roles in the innate immunity against a variety of pathogens. Total RNA from mouse tissues were screened for MBP mRNA by RT-PCR. In addition to liver, S-MBP mRNA was detected in lung, kidney, and testis, and L-MBP mRNA was detected in kidney, thymus, and small intestine. Quantitative RT-PCR revealed that the small intestine is a predominant site of extrahepatic expression of L-MBP. Western blotting with polyclonal Abs against rat L-MBP demonstrated this protein in Triton X-100 extracts of the small intestine obtained from mice that had undergone systemic perfusion. Immunohistochemical staining with an mAb against mouse L-MBP and in situ hybridization revealed that L-MBP is selectively expressed in some villous epithelial cells of the small intestine. These findings suggest that L-MBP plays a role in mucosal innate immunity.