Effects of TGF-β1 on the proliferation and differentiation of human periodontal ligament cells and a human periodontal ligament stem/progenitor cell line

Periodontal ligament (PDL) is a specialized connective tissue that influences the lifespan of the tooth. Transforming growth factor-β1 (TGF-β1) is a multifunctional cytokine, but little is known about the effects of TGF-β1 on PDL cells. Our aim has been to demonstrate the expression of TGF-β1 in rat PDL tissues and to evaluate its effects on the proliferation and gene expression in human PDL cells (HPLCs) and a human PDL stem/progenitor cell line, line 1-11, that we have recently developed. The expression of TGF-β1 in the entire PDL tissue was confirmed immunohistochemically, and both HPLCs and cell line 1-11 expressed mRNA from the TGF-β1, TGF-β type I receptor, and TGF-β type II receptor genes. Although exogenous TGF-β1 stimulated the proliferation of HPLCs, it did not upregulate the expression of alpha-smooth muscle actin (α-SMA), type I collagen (Col I), or fibrillin-1 (FBN1) mRNA or of α-SMA protein in HPLCs, whereas expression for these genes was attenuated by an anti-TGF-β1 neutralizing antibody. In contrast, exogenous TGF-β1 reduced the proliferation of cell line 1-11, although it upregulated the expression of α-SMA, Col I, and FBN1 mRNA and of α-SMA protein in this cell line. In addition, interleukin-1 beta stimulation significantly reduced the expression of TGF-β1 mRNA and protein in HPLCs. Thus, TGF-β1 seems to play an important role in inducing fibroblastic differentiation of PDL stem/progenitor cells and in maintaining the PDL apparatus under physiological conditions.     

Classification of genus Pseudomonas by MALDI-TOF MS based on ribosomal protein coding in S10-spc-alpha operon at strain level

We have proposed a rapid phylogenetic classification at the strain level by MALDI-TOF MS using ribosomal protein matching profiling. In this study, the S10-spc-alpha operon, encoding half of the ribosomal subunit proteins and highly conserved in eubacterial genomes, was selected for construction of the ribosomal protein database as biomarkers for bacterial identification by MALDI-TOF MS analysis to establish a more reliable phylogenetic classification. Our method revealed that the 14 reliable and reproducible ribosomal subunit proteins with less than m/z 15,000, except for L14, coded in the S10-spc-alpha operon were significantly useful biomarkers for bacterial classification at species and strain levels by MALDI-TOF MS analysis of genus Pseudomonas strains. The obtained phylogenetic tree was consisted with that based on genetic sequence (gyrB). Since S10-spc-alpha operons of genus Pseudomonas strains were sequenced using specific primers designed based on nucleotide sequences of genome-sequenced strains, the ribosomal subunit proteins encoded in S10-spc-alpha operon were suitable biomarkers for construction and correction of the database. MALDI-TOF MS analysis using these 14 selected ribosomal proteins is a rapid, efficient, and versatile bacterial identification method with the validation procedure for the obtained results.     

Sharing of Diverse Mycorrhizal and Root-Endophytic Fungi among Plant Species in an Oak-Dominated Cool–Temperate Forest

Most terrestrial plants interact with diverse clades of mycorrhizal and root-endophytic fungi in their roots. Through belowground plant–fungal interactions, dominant plants can benefit by interacting with host-specific mutualistic fungi and proliferate in a community based on positive plant–mutualistic fungal feedback. On the other hand, subordinate plant species may persist in the community by sharing other sets (functional groups) of fungal symbionts with each other. Therefore, revealing how diverse clades of root-associated fungi are differentially hosted by dominant and subordinate plant species is essential for understanding plant community structure and dynamics. Based on 454-pyrosequencing, we determined the community composition of root-associated fungi on 36 co-occurring plant species in an oak-dominated forest in northern Japan and statistically evaluated the host preference phenotypes of diverse mycorrhizal and root-endophytic fungi. An analysis of 278 fungal taxa indicated that an ectomycorrhizal basidiomycete fungus in the genus Lactarius and a possibly endophytic ascomycete fungus in the order Helotiales significantly favored the dominant oak (Quercus) species. In contrast, arbuscular mycorrhizal fungi were generally shared among subordinate plant species. Although fungi with host preferences contributed to the compartmentalization of belowground plant–fungal associations, diverse clades of ectomycorrhizal fungi and possible root endophytes were associated not only with the dominant Quercus but also with the remaining plant species. Our findings suggest that dominant-ectomycorrhizal and subordinate plant species can host different subsets of root-associated fungi, and diverse clades of generalist fungi can counterbalance the compartmentalization of plant–fungal associations. Such insights into the overall structure of belowground plant–fungal associations will help us understand the mechanisms that facilitate the coexistence of plant species in natural communities.     

Caffeine fostering of mycoparasitic fungi against phytopathogens

Caffeine (1,3,7-trimethixanthine) is a typical purine alkaloid produced in more than 80 plant species. Its biological role is considered to strengthen plant''s defense capabilities, directly as a toxicant to biotic attackers (allelopathy) and indirectly as an activator of defense system (priming). Caffeine is actively secreted into rhizosphere through primary root, and possibly affects the structure of microbe community nearby. The fungal community in coffee plant rhizosphere is enriched with particular species, including Trichoderma family, a mycoparasite that attacks and kills phytopathogens by coiling and destroying their hyphae. In the present study, the caffeine response of 8 filamentous fungi, 4 mycoparasitic Trichoderma, and 4 prey phytopathogens, was examined. Results showed that allelopathic effect of caffeine on fungal growth and development was differential, being stronger on pathogens than on Trichoderma species. Upon confronting, the prey immediately ceased the growth, whereas the predator continued to grow, indicating active mycoparasitism to have occurred. Caffeine enhanced mycoparasitism up to 1.7-fold. Caffeine thus functions in a double-track manner against fungal pathogens: first by direct suppression of growth and development, and second by assisting their natural enemy. These observations suggest that caffeine is a powerful weapon in the arms race between plants and pathogens by fostering enemy''s enemy, and we propose the idea of "caffeine fostering" as the third role of caffeine.     

Effects of initial settings on computational protein–ligand docking accuracies for several docking programs

In this study, the influences of initial settings, i.e. initial conformations, configurations and docking parameters, on docking results were investigated. The conformations used in the study were generated by the CAMDAS program. After the conformational search calculations, five structures were selected from the conformer groups according to their conformation energies and root mean square deviations against crystal structures; for example, the lowest energy conformer, as well as the closest and farthest conformers to the crystal structure, was retrieved. Several docking parameter settings were used (default, high speed, generating 50 poses). In this study, docking calculations were conducted using the GOLD, eHiTS, AutoDock, AutoDock vina, FRED and DOCK programs. The success rates of GOLD, eHiTS and FRED were better than those of AutoDock, AutoDock vina and DOCK. The docking results using the farthest conformations were worse than those obtained using other conformations, indicating that some conformation search for the ligand molecule should be performed before the docking calculations.     

How are plant and fungal communities linked to each other in belowground ecosystems? A massively parallel pyrosequencing analysis of the association specificity of root-associated fungi and their host plants

In natural forests, hundreds of fungal species colonize plant roots. The preference or specificity for partners in these symbiotic relationships is a key to understanding how the community structures of root‐associated fungi and their host plants influence each other. In an oak‐dominated forest in Japan, we investigated the root‐associated fungal community based on a pyrosequencing analysis of the roots of 33 plant species. Of the 387 fungal taxa observed, 153 (39.5%) were identified on at least two plant species. Although many mycorrhizal and root‐endophytic fungi are shared between the plant species, the five most common plant species in the community had specificity in their association with fungal taxa. Likewise, fungi displayed remarkable variation in their association specificity for plants even within the same phylogenetic or ecological groups. For example, some fungi in the ectomycorrhizal family Russulaceae were detected almost exclusively on specific oak (Quercus) species, whereas other Russulaceae fungi were found even on “non‐ectomycorrhizal” plants (e.g., Lyonia and Ilex). Putatively endophytic ascomycetes in the orders Helotiales and Chaetothyriales also displayed variation in their association specificity and many of them were shared among plant species as major symbionts. These results suggest that the entire structure of belowground plant–fungal associations is described neither by the random sharing of hosts/symbionts nor by complete compartmentalization by mycorrhizal type. Rather, the colonization of multiple types of mycorrhizal fungi on the same plant species and the prevalence of diverse root‐endophytic fungi may be important features of belowground linkage between plant and fungal communities.     

H-Ferritin Is Preferentially Incorporated by Human Erythroid Cells through Transferrin Receptor 1 in a Threshold-Dependent Manner

Ferritin is an iron-storage protein composed of different ratios of 24 light (L) and heavy (H) subunits. The serum level of ferritin is a clinical marker of the body’s iron level. Transferrin receptor (TFR)1 is the receptor not only for transferrin but also for H-ferritin, but how it binds two different ligands and the blood cell types that preferentially incorporate H-ferritin remain unknown. To address these questions, we investigated hematopoietic cell-specific ferritin uptake by flow cytometry. Alexa Fluor 488-labeled H-ferritin was preferentially incorporated by erythroid cells among various hematopoietic cell lines examined, and was almost exclusively incorporated by bone marrow erythroblasts among human primary hematopoietic cells of various lineages. H-ferritin uptake by erythroid cells was strongly inhibited by unlabeled H-ferritin but was only partially inhibited by a large excess of holo-transferrin. On the other hand, internalization of labeled holo-transferrin by these cells was not inhibited by H-ferritin. Chinese hamster ovary cells lacking functional endogenous TFR1 but expressing human TFR1 with a mutated RGD sequence, which is required for transferrin binding, efficiently incorporated H-ferritin, indicating that TFR1 has distinct binding sites for H-ferritin and holo-transferrin. H-ferritin uptake by these cells required a threshold level of cell surface TFR1 expression, whereas there was no threshold for holo-transferrin uptake. The requirement for a threshold level of TFR1 expression can explain why among primary human hematopoietic cells, only erythroblasts efficiently take up H-ferritin.     

Hepatic overexpression of a dominant negative form of raptor enhances Akt phosphorylation and restores insulin sensitivity in K/KAy mice

Several serine/threonine kinases reportedly phosphorylate serine residues of IRS-1 and thereby induce insulin resistance. In this study, to investigate the effect of mTOR/raptor on insulin signaling and metabolism in K/KAy mice with genetic obesity-associated insulin resistance, a dominant negative raptor, COOH-terminally deleted raptor (raptor-DeltaC(T)), was overexpressed in the liver via injection of its adenovirus into the circulation. Hepatic raptor-DeltaC(T) expression levels were 1.5- to 4-fold that of endogenously expressed raptor. Glucose tolerance in raptor-DeltaC(T)-overexpressing mice improved significantly compared with that of LacZ-overexpressing mice. Insulin-induced activation of p70S6 kinase (p70(S6k)) was significantly suppressed in the livers of raptor-DeltaC(T) overexpressing mice. In addition, insulin-induced IRS-1, Ser(307), and Ser(636/639) phosphorylations were significantly suppressed in the raptor-DeltaC(T)-overexpressing liver, whereas tyrosine phosphorylation of IRS-1 was increased. PI 3-kinase activation in response to insulin stimulation was increased approximately twofold, and Akt phosphorylation was clearly enhanced under both basal and insulin-stimulated conditions in the livers of raptor-DeltaC(T) mice. Thus, our data indicate that suppression of the mTOR/p70(S6k) pathway leads to improved glucose tolerance in K/KAy mice. These observations may contribute to the development of novel antidiabetic agents.     

Molecular Cloning,Modeling, and Site-Directed Mutagenesis of Type III Polyketide Synthase from <Emphasis Type="Italic">Sargassum binderi</Emphasis> (Phaeophyta)

Type III polyketide synthases (PKSs) produce an array of metabolites with diverse functions. In this study, we have cloned the complete reading frame encoding type III PKS (SbPKS) from a brown seaweed, Sargassum binderi, and characterized the activity of its recombinant protein biochemically. The deduced amino acid sequence of SbPKS is 414 residues in length, sharing a higher sequence similarity with bacterial PKSs (38% identity) than with plant PKSs. The Cys-His-Asn catalytic triad of PKS is conserved in SbPKS with differences in some of the residues lining the active and CoA binding sites. The wild-type SbPKS displayed broad starter substrate specificity to aliphatic long-chain acyl-CoAs (C₆–C₁₄) to produce tri- and tetraketide pyrones. Mutations at H³³¹ and N³⁶⁴ caused complete loss of its activity, thus suggesting that these two residues are the catalytic residues for SbPKS as in other type III PKSs. Furthermore, H227G, H227G/L366V substitutions resulted in increased tetraketide-forming activity, while wild-type SbPKS produces triketide α-pyrone as a major product. On the other hand, mutant H227G/L366V/F93A/V95A demonstrated a dramatic decrease of tetraketide pyrone formation. These observations suggest that His²²⁷ and Leu³⁶⁶ play an important role for the polyketide elongation reaction in SbPKS. The conformational changes in protein structure especially the cavity of the active site may have more significant effect to the activity of SbPKS compared with changes in individual residues.