Reduced pain hypersensitivity and inflammation in mice lacking microsomal prostaglandin e synthase-1

We examined the in vivo role of membrane-bound prostaglandin E synthase (mPGES)-1, a terminal enzyme in the PGE2-biosynthetic pathway, using mPGES-1 knockout (KO) mice. Comparison of PGES activity in the membrane fraction of tissues from mPGES-1 KO and wild-type (WT) mice indicated that mPGES-1 accounted for the majority of lipopolysaccharide (LPS)-inducible PGES in WT mice. LPS-stimulated production of PGE2, but not other PGs, was impaired markedly in mPGES-1-null macrophages, although a low level of cyclooxygenase-2-dependent PGE2 production still remained. Pain nociception, as assessed by the acetic acid writhing response, was reduced significantly in KO mice relative to WT mice. This phenotype was particularly evident when these mice were primed with LPS, where the stretching behavior and the peritoneal PGE2 level of KO mice were far less than those of WT mice. Formation of inflammatory granulation tissue and attendant angiogenesis in the dorsum induced by subcutaneous implantation of a cotton thread were reduced significantly in KO mice compared with WT mice. Moreover, collagen antibody-induced arthritis, a model for human rheumatoid arthritis, was milder in KO mice than in WT mice. Collectively, our present results provide unequivocal evidence that mPGES-1 contributes to the formation of PGE2 involved in pain hypersensitivity and inflammation.     

Identification of beta-tubulin isoform V as an autoantigen in allergic rhinitis by a proteomic approach

Autoantibodies to IgE and beta2-adrenergic receptor have been reported in patients with allergic rhinitis. To investigate whether autoimmunity in allergic rhinitis is directed to such limited molecules or directed to a wide range of self proteins, we here attempted to survey autoantigens/autoantibodies comprehensively, using proteomics. Specifically, we separated proteins extracted from peripheral blood mononuclear cells by 2-dimensional electrophoresis and then detected autoantigens by subsequent western blotting with sera from patients with allergic rhinitis. As a result, we detected multiple autoantigens, some of which were further identified by mass fingerprinting. Next, we confirmed antigenicity of one of the identified autoantigens, beta-tubulin isoform V (beta-tubV), using a recombinant protein and then measured prevalence of the anti-beta-tubV autoantibodies. As a result, 52% of the tested patients with allergic rhinitis were found to possess anti-beta-tubV autoantibodies. Our study indicates that autoimmunity is a common phenomena and beta-tubV is one of the major autoantigens in allergic rhinitis.     

Structural stability of covalently linked GroES heptamer: advantages in the formation of oligomeric structure

In order to understand how inter-subunit association stabilizes oligomeric proteins, a single polypeptide chain variant of heptameric co-chaperonin GroES (tandem GroES) was constructed from Escherichia coli heptameric GroES by linking consecutively the C-terminal of one subunit to the N-terminal of the adjacent subunit with a small linker peptide. The tandem GroES (ESC7) showed properties similar to wild-type GroES in structural aspects and co-chaperonin activity. In unfolding and refolding equilibrium experiments using guanidine hydrochloride (Gdn-HCl) as a denaturant at a low protein concentration (50 microg ml(-1)), ESC7 showed a two-state transition with a greater resistance toward Gdn-HCl denaturation (Cm=1.95 M) compared to wild-type GroES (Cm=1.1 M). ESC7 was found to be about 10 kcal mol(-1) more stable than the wild-type GroES heptamer at 50 microg ml(-1). Kinetic unfolding and refolding experiments of ESC7 revealed that the increased stability was mainly attributed to a slower unfolding rate. Also a transient intermediate was detected in the refolding reaction. Interestingly, at the physiological GroES concentration (>1 mg ml(-1)), the free energy of unfolding for GroES heptamer exceeded that for ESC7. These results showed that at low protein concentrations (<1 mg ml(-1)), the covalent linking of subunits contributes to the stability but also complicates the refolding kinetics. At physiological concentrations of GroES, however, the oligomeric state is energetically preferred and the advantages of covalent linkage are lost. This finding highlights a possible advantage in transitioning from multi-domain proteins to oligomeric proteins with small subunits in order to improve structural and kinetic stabilities.     

The human megakaryocytic cell line UT‐7/TPO expresses functional platelet agonist signals mediated through GPVI and thromboxane receptor

We have demonstrated that a unique megakaryocytic cell line UT‐7/TPO could respond to one of the primary platelet signals through GP (glycoprotein) VI and a secondary signal of the AA (arachidonic acid) cascade. Unlike other megakaryocytic cell lines, UT‐7/TPO was found to express GPVI and its associate signal molecule of FcRγ (Fc receptor γ chain). When UT‐7/TPO was stimulated with the GPVI agonist convulxin, the [Ca²⁺]i (intracellular Ca²⁺) was elevated in a convulxin concentration‐dependent manner, and [Ca²⁺]i elevation was blocked by pretreatment with the Src family kinase inhibitor PP2 and the phospholipase inhibitor U73122. These results strongly indicate that endogenously expressed GPVI signal molecules are functional in UT‐7/TPO. Concerning the AA cascade, the expression of COX (cyclooxygenase)‐1 and TX (thromboxane) synthase was observed, and this cell line was able to produce TX by exogenous AA, followed by [Ca²⁺]i elevation mediated through the TX receptor. It is worth noting that convulxin stimulation did not cause TX generation, even through the GPVI pathway and the AA cascade are functional in this cell line. As there are many reports that convulxin‐stimulated platelets failed to produce TX, it is suggested that UT‐7/TPO has the same property as the platelets in regards to convulxin stimulation. Thus, UT‐7/TPO is useful for the observation of both the GPVI pathway and AA cascade without requiring either the induction of differentiation or GPVI transfection. Furthermore, this cell line provides a new tool for research on platelet activation signals.     

In Situ Microspatial Imaging Using Two-Photon and Confocal Laser Scanning Microscopy of Bacteria and Extracellular Polymeric Secretions (EPS) Within Marine Stromatolites

The combination of a hydrophilic embedding resin, Nanoplast, with fluorescent probes, and subsequent imaging using two-photon and confocal laser scanning microscopy (2P-LSM and CLSM) has allowed in imaging of the in situ microspatial arrangements of microbial cells and their extracellular polymeric secretion (EPS) within marine stromatolites. Optical sectioning by 2P-LSM and CLSM allowed imaging of endolithic cyanobacteria cells, Solentia sp., seen within carbonate sand grains. 2P-LSM allowed very clear imaging with a high resolution of bacteria using DAPI, which normally require UV excitation and reduced photo-bleaching of fluorescent probes.