Inhibition of activation of human peripheral blood eosinophils by Y-24180, an antagonist to platelet-activating factor receptor.

We examined the effects of Y-24180, a potent and long-acting antagonist to platelet-activating factor (PAF) receptor, on the PAF- or leukotriene B4 (LTB4)-induced activation of eosinophils using human peripheral blood in vitro. As activation markers, CD11b expression level and soluble intercellular adhesion molecule-1 (sICAM-1)-binding activity were analyzed by flow cytometry. Y-24180 significantly inhibited PAF-induced increase in the ratio of strongly positive cells for CD11b expression and sICAM-1 binding at 0.01 microM or more. WEB 2086, another PAF receptor antagonist, also inhibited the increase significantly at 1 microM or more. LTB4-induced increases in the ratio of strongly positive cells for CD11b expression and sICAM-1 binding were inhibited by Y-24180 at 1 microM, but not WEB 2086 up to 10 microM. These results indicate that Y-24180 inhibits the PAF- or LTB4-induced activation of eosinophils in human peripheral blood more potently than WEB 2086.     

Distribution of Rho-kinase in the bovine brain.

Rho-associated kinase (Rho-kinase) is a serine/threonine protein kinase downstream of the small GTPase Rho, which participates in signaling pathways of many cellular functions. Although Rho-kinase is implicated in the regulation of the morphology of neuronal cells, the distribution of Rho-kinase in the brain has not been elucidated yet. In this study, we investigated the distribution of Rho-kinase using three antibodies recognizing the different epitopes of Rho-kinase. Rho-kinase was abundantly expressed in the gray matter in comparison with the white matter. Strong immunoreactivity was observed in the pyramidal neurons of the cerebral cortex and hippocampus and in the Purkinje cells of the cerebellum. These results indicate that Rho-kinase is abundantly distributed in neurons and might play an important role in remodeling of neurites.     

Specific interactions between Porphyromonas gingivalis fimbriae and human extracellular matrix proteins

The interactions of the extracellular matrix (ECM) proteins (laminin, elastin, fibronectin, type I collagen, thrombospondin and vitronectin) with the fimbriae of Porphyromonas gingivalis were analyzed based on surface plasmon resonance (SPR) spectroscopy using a biomolecular interaction analyzing system (BIAcore). The BIAcore profiles demonstrated that fimbriae specifically bound to all of the ECM proteins with significant association constants (Ka). Vitronectin showed the highest affinity to fimbriae (Ka = 3.79 x 10(6) M-1), while the affinity of laminin was lowest (Ka = 2.15 x 10(6) M-1). A synthetic peptide which is a potent inhibitor of fimbrial binding to salivary proteins was not significantly effective on the fimbrial interactions with the ECM proteins. Using polystyrene microtiter plates revealed that P. gingivalis fimbriae bound markedly to immobilized fibronectin and type I collagen, while the interaction of fimbriae with the other ECM proteins was not clearly demonstrated. These results suggest that interactions between fimbriae and the ECM proteins occur with specific affinities which are not mediated by mechanisms identical to those of salivary proteins. It was also shown that SPR spectroscopy is a useful method to analyze these specific interactions.     

DNA recognition by β-sheets

The modes of DNA recognition by β-sheets are analyzed by using the known crystal and solution three-dimensional structures of DNA-protein complexes. Close fitting of the protein surface and the DNA surface determines the binding geometry. Interaction takes place so that essentially the N-to-C direction of the β-strands either follows or crosses the DNA groove. Upon following the major groove a two-stranded antiparallel β-sheet dives into the groove and contacts DNA bases with its convex side facing the DNA, while upon following the minor groove, it binds around the sugar-phosphate backbones, with its opposite concave side shielding the DNA. In order for the β-strands crossing the minor groove to interact with the DNA, the dinucleotide steps need to almost totally helically untwist and roll around major groove. The β-sheet, on the other hand, needs to adopt a concave curvature on the binding surface in the direction that follows the DNA minor groove, and a convex surface in the direction that bridges the sugar-phosphate backbones across the groove. The result is to produce a hyperbolic paraboloidal DNA-binding surface. © 1998 John Wiley & Sons, Inc. Biopoly 44: 335–359, 1997     

Potent antiobesity effect of a short-length peptide YY-analogue continuously administered in mice

The gastrointestinal peptide, peptide YY_3–36 (PYY_3–36) and its shorter peptide analogues have been reported to reduce appetite by activating the neuropeptide Y2 receptor (Y2R), which is associated with obesity and other metabolic diseases. A 14-amino acid PYY analogue, Ac-[d-Pro²⁴,Cha^27,28,36,Aib³¹]PYY(23–36) (3), showed high binding affinity and agonist activity for the Y2R, similar to that of PYY_3–36, but had weak anorectic activity upon continuous administration in lean mice. Three amino acid substitutions [Pya(4)²⁶, Aib²⁸, Lys³⁰], which contributed to the decreased hydrophobicity of 3, efficiently increased its anorectic activity. The compound containing these three amino acids, Ac-[d-Pro²⁴,Pya(4)²⁶,Cha^27,36,Aib^28,31,Lys³⁰]PYY(23–36) (22), exerted more potent and durable food intake suppression than that by PYY_3–36 in lean mice, as well as excellent Y2R agonist activity (EC₅₀: 0.20 nM) and good subcutaneous bioavailability (66.6%). The 11-day continuous administration of 22 at 1 mg/kg/day successfully produced antiobese and antidiabetic effects, with more than 20% body weight loss in obese and Type 2 diabetes ob/ob model mice.     

The Succession of Bacterial Community Structure in Groundwater from a 250-m Gallery in the Horonobe Underground Research Laboratory

We investigated the change in bacterial community structure after drilling boreholes, 09-V250-M02 and 09-V250-M03, in the 250-m deep research gallery of the Horonobe Underground Research Laboratory. In the 09-V250-M02 borehole, ?-Proteobacteria were predominantly detected in the clone library analyses of the groundwater samples conducted immediately after drilling. All the ?-Proteobacteria clones were closely related to Arcobacter spp., which are known to be sulfide-oxidizing chemoautotrophic bacteria. After 4 years, the microbial structure drastically changed, and most detected operational taxonomic units were uncultured species such as candidate division OP9 and Chloroflexi relatives, which are frequently detected in deep sea sediments. The results indicated that the microbial community structure was drastically affected by borehole drilling and was concomitant with oxidation perturbation. However, these disturbed microbial communities changed within a few years to a microbial community composed of uncultivated species such as OP9 and Chloroflexi.     

A Round-bottom 96-well Polystyrene Plate Coated with 2-methacryloyloxyethyl Phosphorylcholine as an Effective Tool for Embryoid Body Formation

In this study, we proposed a culture method for forming embryoid bodies (EBs) from mouse embryonic stem (ES) cells using a round-bottom 96-well polystyrene plate coated with 2-methacryloyloxyethyl phosphorylcholine (MPC plate). MPC is a phospholipid biocompatible polymer and prevents cells from adhering to the culture surface. The ES cells were seeded at 1000 cells per well in the MPC plate with 200 μl of medium. After 5 days of static incubation, a spherical cell aggregate termed EB was formed in a well. The size (diameter) of resulting EB was approximately 550 μm and it contained approx. 22,000 cells. It seems that the non-adhesiveness and the roundness of the well are important factors to form a good EB. Transferring the EBs to the attached differentiation culture, the EBs spread out and flattened, and the beating cells (cardiomyocytes) were effectively generated in the outgrowth of EBs. The round-bottom 96-well polystyrene plate coated with MPC is an effective tool for EB formation.     

P. gingivalis accelerates gingival epithelial cell progression through the cell cycle

P. gingivalis, an opportunistic pathogen in periodontal disease, can reside within the epithelial cells that line the gingival crevice. A proteomic analysis revealed that infection of gingival epithelial cells with P. gingivalis induces broadly based changes in the level and phosphorylation status of proteins that exert multi-level control on the eukaryotic cell cycle. Pathways that were impacted by P. gingivalis included those involving cyclins, p53 and PI3K. The predicted infection-dependent phenotype was confirmed by cytofluorimetry that showed an enhanced proliferation rate of gingival epithelial cells infected with P. gingivalis associated with accelerated progression through the S-phase. Elevated cell proliferation was dependent on the presence of the long fimbriae of P. gingivalis. The ability of P. gingivalis, a common inhabitant of the subgingival crevice, to accelerate cell cycling could have biological consequences for barrier and signaling functions, and for physiological status, of the gingival epithelium.     

Prolactin induces MFG-E8 production in macrophages via transcription factor C/EBPβ-dependent pathway

The lactogenic hormone prolactin (PRL) regulates milk protein gene expression in mammary glands. To maintain homeostatic balance in the body, milk fat globule epidermal growth factor 8 (MFG-E8) is vital for phagocytic clearance of apoptotic cells. We investigated the effects of PRL on MFG-E8 expression in macrophages by evaluating its promoter function. Macrophages were stimulated with PRL, and the expression of MFG-E8 was determined using real-time PCR and Western blotting. The role of MFG-E8 on phagocytosis of apoptotic cells in PRL-treated macrophages was assessed using microscopy, while the response of PRL to MFG-E8 expression was evaluated using luciferase assay. Following treatment with PRL, significant up-regulations of the PRL receptor and MFG-E8 were observed in macrophages, though PRL-treated macrophages more efficiently engulfed apoptotic cells. The results of MFG-E8 promoter analysis showed considerable up-regulation of promoter activity in macrophages following PRL treatment and results from mutation analysis of the MFG-E8 promoter suggested that the C/EBPβ binding site was responsible for PRL-induced activation of the MFG-E8 promoter. C/EBPβ activity was found to be up-regulated in PRL-treated cells as revealed by an electrophoretic mobility shift assay (EMSA). In conclusion, PRL is a potent inducer of MFG-E8 expression in macrophages, while its effect is mediated by the presence of a responsive element in the MFG-E8 promoter.     

Molecular structure and properties of lectin from tomato fruit

A cDNA encoding tomato fruit lectin was cloned from an unripe cherry-tomato fruit cDNA library. The isolated lectin cDNA contained an open reading frame encoding 365 amino acids, including peptides that were sequenced. The deduced sequence consisted of three distinct domains: (i) an N-terminal short extensin-like domain; (ii) a Cys-rich carbohydrate binding domain composed of four almost identical chitin-binding domains; (iii) an internal extensin-like domain of 101 residues containing 15 SerPro(4) motifs inserted between the first and second chitin-binding domains. The molecular weight of the lectin was 65,633 and that of the deglycosylated lectin was 32,948, as determined by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). This correlated with the estimated molecular weight of the deduced sequence. Recombinant tomato lectin expressed in Pichia pastoris possessed chitin-binding but not hemagglutinating activity. These findings confirmed that the cDNA encoded tomato lectin.