Evaluation of seasonal dynamics of fungal DNA assemblages in a flow-regulated stream in a restored forest using eDNA metabarcoding

Investigation of seasonal variation in fungal communities is essential for understanding biodiversity and ecosystem functions. However, the conventional sampling method, with substrate removal and high spatial heterogeneity of community composition, makes surveying the seasonality of fungal communities challenging. Recently, water environmental DNA (eDNA) analysis has been explored for its utility in biodiversity surveys. In this study, we assessed whether the seasonality of fungal communities can be detected by monitoring eDNA in a forest stream. We conducted monthly water sampling in a forest stream over 2 years and used DNA metabarcoding to identify fungal eDNA. The stream water contained DNA from functionally diverse aquatic and terrestrial fungi, such as plant decomposers, parasites and mutualists. The variation in the fungal assemblage showed a regular annual periodicity, meaning that the assemblages in a given season were similar, irrespective of the year or sampling. Furthermore, the strength of the annual periodicity varied among functional groups. Our results suggest that forest streams may act as a ‘trap’ for terrestrial fungal DNA derived from different habitats, allowing the analysis of fungal DNA in stream water to provide information about the temporal variation in fungal communities in both the aquatic and the surrounding terrestrial ecosystems.     

Alendronate suppresses tumor angiogenesis by inhibiting Rho activation of endothelial cells

We previously reported that alendronate inhibits intraperitoneal dissemination in an in vivo ovarian cancer model. Recently, nitrogen-containing bisphosphonates have been reported to have antiangiogenic activities. In this study, alendronate inhibited human umbilical vein endothelial cell (HUVEC) migration and capillary-like structure formation in vitro. These inhibitory effects were associated with reduced Rho activation and suppression of the formation of actin stress fibers and focal adhesions in HUVECs. Furthermore, the inhibition by alendronate was reversed by geranylgeraniol, which abrogated the inhibition of Rho geranylgeranylation. Next, we examined the effect of alendronate on angiogenesis in disseminated ovarian tumors of athymic immunodeficient mice. Alendronate treatment reduced the intra-tumor neoangiogenesis compared with that in the non-treated mice, although tumor-derived VEGF expression was not altered. In conclusion, the in vivo anti-tumor effect of alendronate might be derived, at least in part, from its direct antiangiogenic effects on intra-tumor endothelial cells by inhibiting Rho geranylgeranylation.     

Induction of ovulation, mating, and conception in androgen-treated immature rats

Attempts were made to induce pregnancy in androgen-treated immature rats. Treatment with PMSG alone, which causes ovulation in normal immature rats, failed to cause ovulation in androgenized rats. However, treatment with PMSG plus LHRH was effective in causing ovulation. After ovulation, some of the normal and androgenized rats mated. Normal mated rats became pregnant but androgenized mated rats did not. However, when a pituitary gland was transplanted from a normal rat into the kidney capsule of an androgenized rat to maintain functional corpora lutea, implantation occurred in some of the mated animals. The positive decidual reaction in the uteri of such androgenized rats was similar to that observed in normal rats. These results suggest that the uterine sensitivity to blastocyst implantation of androgenized immature rats may be normal.     

The PWWP domain of mammalian DNA methyltransferase Dnmt3b defines a new family of DNA-binding folds

The PWWP domain is a weakly conserved sequence motif found in > 60 eukaryotic proteins, including the mammalian DNA methyltransferases Dnmt3a and Dnmt3b. These proteins often contain other chromatin-association domains. A 135-residue PWWP domain from mouse Dnmt3b (amino acids 223--357) has been structurally characterized at 1.8 A resolution. The N-terminal half of this domain resembles a barrel-like five-stranded structure, whereas the C-terminal half contains a five-helix bundle. The two halves are packed against each other to form a single structural module that exhibits a prominent positive electrostatic potential. The PWWP domain alone binds DNA in vitro, probably through its basic surface. We also show that recombinant Dnmt3b2 protein (a splice variant of Dnmt3b) and two N-terminal deletion mutants (Delta218 and Delta369) have approximately equal methyl transfer activity on unmethylated and hemimethylated CpG-containing oligonucleotides. The Delta218 protein, which includes the PWWP domain, binds DNA more strongly than Delta369, which lacks the PWWP domain.     

Crystal structures of bacterial lipoprotein localization factors,LolA and LolB

Lipoproteins having a lipid-modified cysteine at the N-terminus are localized on either the inner or the outer membrane of Escherichia coli depending on the residue at position 2. Five Lol proteins involved in the sorting and membrane localization of lipoprotein are highly conserved in Gram-negative bacteria. We determined the crystal structures of a periplasmic chaperone, LolA, and an outer membrane lipoprotein receptor, LolB. Despite their dissimilar amino acid sequences, the structures of LolA and LolB are strikingly similar to each other. Both have a hydrophobic cavity consisting of an unclosed beta barrel and an alpha-helical lid. The cavity represents a possible binding site for the lipid moiety of lipoproteins. Detailed structural differences between the two proteins provide significant insights into the molecular mechanisms underlying the energy-independent transfer of lipoproteins from LolA to LolB and from LolB to the outer membrane. Furthermore, the structures of both LolA and LolB determined from different crystal forms revealed the distinct structural dynamics regarding the association and dissociation of lipoproteins. The results are discussed in the context of the current model for the lipoprotein transfer from the inner to the outer membrane through a hydrophilic environment.     

Localization of 9- and 13-oxo-octadecadienoic acids in tomato fruit

We previously reported that the two peroxisome proliferator-activated receptor-α agonists, 9- and 13-oxo-octadecadienoic acids (oxo-ODAs), were found in the tomato fruit. However, their localization remains unknown. Herein, we showed that oxo-ODAs localize primarily in the fruit peel and their amount increases after the homogenization of the tomato fruit.     

S-Carboxymethylcysteine Synthase from Escherichia coli

An enzyme that catalyzes the synthesis of S-carboxymethyl- l-cysteine from 3-chloro- l-alanine (3-Cl-Ala) and thioglycolic acid was found in Escherichia coli W3110 and was designated as S- carboxymethyl-l-cysteine synthase. It was purified from the cell-free extract to electrophoretic homogeneity and was crystallized. The enzyme has a molecular weight of 84,000 and gave one band corresponding to a molecular weight of 37,000 on SDS-polyacrylamide gel electrophoresis. The purified enzyme catalyzed the β-replacement reactions between 3-CI-AIa and various thiol compounds. The apparent Km values for 3-Cl-Ala and thioglycolic acid were 40 mM and 15.4 mM. The enzyme showed very low activity as to the α,β-elimination reaction with 3-Cl-Ala and l-serine. It was not inactivated on the incubation with 3-Cl-Ala. The absorption spectrum of the enzyme shows a maximum at 412 nm, indicating that it contains pyridoxal phosphate as a cofactor. The N-terminal amino acid sequence was determined and the corresponding sequence was detected in the protein sequence data bank, but no homogeneous sequence was found.     

Activation of detergent-solubilized rat hepatic 5'-iodothyronine deiodinase by NADPH and nonglutathione cytosolic components

An investigation was made of the possible role of the hepatic microsomal membrane in the activation of 5'-iodothyronine deiodinase (5'-DI) by a cytosolic activating system consisting of fraction A (relative mass (Mr) greater than 60,000), fraction B (Mr, approximately 13,000), and NADPH. Activation of 5'-DI in washed microsomes was compared with that of a microsome extract prepared by solubilization with 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulphonate and further purification by fractional precipitation with polyethylene glycol and by DEAE-Sephacel chromatography. All 5'-DI preparations exhibited qualitatively similar dependence upon NADPH and cytosolic factors in fractions A and B for 5'-DI activation and were relatively unresponsive to NADH. Activation of solubilized preparations, unlike that of intact microsomes, was more readily inhibited by low concentrations of detergent and not inhibited by NADPH concentrations above 0.25 mM. Attempted purification of 5'-DI failed to produce a substantial increase in specific activity of the enzyme. It is concluded that, while glutathione-independent cytosolic factors and NADPH can activate 5'-DI in the absence of an intact microsomal membrane, some membrane constituents removed during solubilization and purification of the enzyme are required for maximal activation.