Different initial responses of the canopy herbivory rate in mature oak trees to experimental soil and branch warming in a soil‐freezing area

Plants and insects comprise more than 50% of known species on earth, and their interactions are of major importance in most natural ecosystems. To understand the mechanisms by which global warming affects plant–insect interactions in the canopy of mature cool‐temperate forests with a freeze–thaw cycle, we examined changes in the herbivory rate and leaf traits in oak Quercus crispula. From 2007 to 2009, we experimentally increased the temperature of the surrounding soil and canopy branches of mature oak trees by approximately 5°C using electric heating cables. Soil warming decreased the rate of herbivory in the canopy, whereas branch warming had no effect. The magnitude of the effect of soil warming on canopy herbivory varied. For the first year, the decrease was 32%, but this doubled (63%) in the third year. Branch warming did not affect canopy leaf traits; however, soil warming decreased the leaf nutritional quality by decreasing N and increasing the carbon:nitrogen (CN) ratio for three years. Additionally, soil warming increased total phenolics in the third year. Stepwise multiple regression models showed that among the leaf traits that were changed by soil warming, N explained the variation in herbivory for the first and second years, whereas total phenolics explained it for the third year. Our experimental results demonstrate that soil warming drives the rate of herbivory in the canopy of mature oak trees, and the magnitude of the soil warming effect was gradually enhanced during the initial three years. This suggests the importance of belowground temperature elevation in predicting the effect of global warming on plant–insect interactions in a forest canopy.     

Anatomical patterns of condensed tannin in fine roots of tree species from a cool-temperate forest

Background and AimsCondensed tannin (CT) is an important compound in plant biological structural defence and for tolerance of herbivory and environmental stress. However, little is known of the role and location of CT within the fine roots of woody plants. To understand the role of CT in fine roots across diverse species of woody dicot, we evaluated the localization of CT that accumulated in root tissue, and examined its relationships with the stele and cortex tissue in cross-sections of roots in 20 tree species forming different microbial symbiotic groups (ectomycorrhiza and arbuscular mycorrhiza).MethodsIn a cool-temperate forest in Japan, cross-sections of sampled roots in different branching order classes, namely, first order, second to third order, fourth order, and higher than fourth order (higher order), were measured in terms of the length-based ratios of stele diameter and cortex thickness to root diameter. All root samples were then stained with ρ-dimethylaminocinnamaldehyde solution and we determined the ratio of localized CT accumulation area to the root cross-section area (CT ratio).Key ResultsStele ratio tended to increase with increasing root order, whereas cortex ratio either remained unchanged or decreased with increasing order in all species. The CT ratio was significantly positively correlated to the stele ratio and negatively correlated to the cortex ratio in second- to fourth-order roots across species during the shift from primary to secondary root growth. Ectomycorrhiza-associated species mostly had a higher stele ratio and lower cortex ratio than arbuscular mycorrhiza-associated species across root orders. Compared with arbuscular mycorrhiza species, there was greater accumulation of CT in response to changes in the root order of ectomycorrhiza species.ConclusionsDifferent development patterns of the stele, cortex and CT accumulation along the transition from root tip to secondary roots could be distinguished between different mycorrhizal associations. The CT in tissues in different mycorrhizal associations could help with root protection in specific branching orders during shifts in stele and cortex development before and during cork layer formation.     

An efficient and practical method for solid-phase synthesis of tripeptide-bearing glycopeptide antibiotics: combinatorial parallel synthesis of carboxamide derivatives of chloroorienticin B

An efficient and practical method was established for solid-phase parallel synthesis of the peptide-bearing carboxamide derivatives of chloroorienticin B, and over 80 compounds were synthesized simultaneously. Among the derivatives prepared, compounds having both tryptophan and tyrosine residues (1-3) were found to possess potent antibacterial activity against VRE.     

Roles of salivary components in Streptococcus mutans colonization in a new animal model using NOD/SCID.e2f1-/- mice

Streptococcus mutans plays an important role in biofilm formation on the tooth surface and is the primary causative agent of dental caries. The binding of S. mutans to the salivary pellicle is of considerable etiologic significance and is important in biofilm development. Recently, we produced NOD/SCID.e2f1(-/-) mice that show hyposalivation, lower salivary antibody, and an extended life span compared to the parent strain: NOD.e2f1(-/-). In this study we used NOD/SCID.e2f1(-/-) 4 or 6 mice to determine the roles of several salivary components in S. mutans colonization in vivo. S. mutans colonization in NOD/SCID.e2f1(-/-) mice was significantly increased when mice were pre-treated with human saliva or commercial salivary components. Interestingly, pre-treatment with secretory IgA (sIgA) at physiological concentrations promoted significant colonization of S. mutans compared with sIgA at higher concentrations, or with human saliva or other components. Our data suggest the principal effects of specific sIgA on S. mutans occur during S. mutans colonization, where the appropriate concentration of specific sIgA may serve as an anti-microbial agent, agglutinin, or an adherence receptor to surface antigens. Further, specific sIgA supported biofilm formation when the mice were supplied 1% sucrose water and a non-sucrose diet. The data suggests that there are multiple effects exerted by sIgA in S. mutans colonization, with synergistic effects evident under the condition of sIgA and limited nutrients on colonization in NOD/SCID.e2f1(-/-) mice. This is a new animal model that can be used to assess prevention methods for dental biofilm-dependent diseases such as dental caries.     

Advanced method for high-throughput expression of mutated eukaryotic membrane proteins in Saccharomyces cerevisiae

Crystallization of eukaryotic membrane proteins is a challenging, iterative process. The protein of interest is often modified in an attempt to improve crystallization and diffraction results. To accelerate this process, we took advantage of a GFP-fusion yeast expression system that uses PCR to direct homologous recombination and gene cloning. We explored the possibility of employing more than one PCR fragment to introduce various mutations in a single step, and found that when up to five PCR fragments were co-transformed into yeast, the recombination frequency was maintained as the number of fragments was increased. All transformants expressed the model membrane protein, while the resulting plasmid from each clone contained the designed mutations only. Thus, we have demonstrated a technique allowing the expression of mutant membrane proteins within 5 days, combining a GFP-fusion expression system and yeast homologous recombination.     

The amino acids involved in the distinct carbohydrate specificities between macrophage galactose-type C-type lectins 1 and 2 (CD301a and b) of mice

Binding specificities of mouse macrophage galactose-type C-type lectin 1 (MGL1/CD301a) and 2 (MGL2/CD301b) toward various oligosaccharides were compared by frontal affinity chromatography. MGL1 preferentially bound oligosaccharides containing Lewis(X) (Le(X)) trisaccharides among 111 oligosaccharides tested, whereas MGL2 preferentially bound globoside Gb4. The important amino acids for the preferential bindings were investigated by pair-wise site-directed mutagenesis at positions 61, 89, 97, 100, 110-113, 115, 124, and 125 in the soluble recombinant carbohydrate recognition domains (CRD) prepared in Escherichia coli and purified with galactose-Sepharose. Mutations of Val, Ala, Thr, and Phe at positions 61, 89, 111 and 125 on MGL1 CRD caused reductions in Le(X) binding. Mutations of MGL2 CRD at Leu, Arg, Arg, and Tyr at positions 61, 89, 115 and 125 were implicated in the preference for beta-GalNAc. Le(X) binding was observed with MGL2 mutants of Arg89Ala and Arg89Ala/Ser111Thr. MGL1 mutants of Ala89Arg and Ala89Arg/Pro115Arg showed beta-GalNAc bindings. Molecular modeling illustrated potential direct molecular interactions of Leu61, Arg89, and His109 in MGL2 CRD with GalNAc.     

Matrix mineralization as a trigger for osteocyte maturation.

The morphology of the osteocyte changes during the cell's lifetime. Shortly after becoming buried in the matrix, an osteocyte is plump with a rich rough endoplasmic reticulum and a well-developed Golgi complex. This "immature" osteocyte reduces its number of organelles to become a "mature" osteocyte when it comes to reside deeper in the bone matrix. We hypothesized that mineralization of the surrounding matrix is the trigger for osteocyte maturation. To verify this, we prevented mineralization of newly formed matrix by administration of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) and then examined the morphological changes in the osteocytes in rats. In the HEBP group, matrix mineralization was disturbed, but matrix formation was not affected. The osteocytes found in the unmineralized matrix were immature. Mature osteocytes were seen in the corresponding mineralized matrix in the control group. The immature osteocytes in the unmineralized matrix failed to show immunoreactivity with anti-sclerostin antibody, whereas mature osteocytes in the mineralized matrix showed immunoreactivity in both control and HEBP groups. These findings suggest that mineralization of the matrix surrounding the osteocyte is the trigger for cytodifferentiation from a plump immature form to a mature osteocyte. The osteocyte appears to start secreting sclerostin only after it matures in the mineralized bone matrix.     

Identification of ACE-inhibitory peptides in salt-free soy sauce that are transportable across caco-2 cell monolayers

In present study, we aimed to identify angiotensin I-converting enzyme (ACE)-inhibitory peptides from a salt-free soy sauce (SFS), a newly developed antihypertensive seasoning obtained by Aspergillus oryzae fermentation of soybean in the absence of salt, which can be transported through caco-2 cell monolayers. Through an Ussing transport investigation of SFS across caco-2 cell monolayers, three di-peptides, Ala-Phe, Phe-Ile and Ile-Phe, were successfully identified from the SFS as transportable inhibitory peptides. Ala-Phe and Ile-Phe, but not Phe-Ile, exhibited ACE-inhibitory activity with IC(50) values of 165.3 microM and 65.8 microM, respectively. Kinetic studies revealed that Ile-Phe (Km: 3.1 mM, P(app): 2.4 x 10(-6) cm/s) exhibited greater affinity toward the transport compared with Ala-Phe (K(m): 48.1 mM, P(app): 1.4 x 10(-6) cm/s) and Phe-Ile (K(m): 12.7 mM, P(app): 1.4 x 10(-6) cm/s).