IgE crosslinkage of Fcepsilon receptor I induces both production and activation of matrix metalloproteinase-9 in mast cells

Since mast cells play pivotal roles in allergic inflammations, we investigated how IgE-mediated stimulation modulated mast cell matrix metalloproteinase (MMP)-9 production, and its enzymatic activation. In this study, we clearly demonstrated that proMMP-9 released from murine bone marrow-derived cultured mast cells (BMCMC) was activated to its valid form after crosslinking of surface immunoglobulin (Ig)E. Serine protease inhibitors sensitive to chymases inhibited the phenomenon, indicating that certain chymases may be responsible for activation of proMMP-9. Although binding of IgE to its specific receptors did not alter MMP-9 production, the IgE crosslinkage increased both expression of mRNA, and production of MMP-9 in mast cells. Glucocorticoid declined extra cellular processing of proMMP-9 without affecting mRNA expression. These findings give rise to the possibility that production and activation of mast cell MMP-9 may be increased in the affected sites, thereby resulting in an exacerbation of tissue degradation in inflammatory conditions.     

Simultaneous fluorometric measurement of histamine and tele-methylhistamine levels in rodent brain by high-performance liquid chromatography

An improved high-performance liquid chromatography (HPLC) method was developed for simultaneous analysis of histamine (HA) and tele-methylhistamine (tele-MHA) levels in mouse and rat brain. The method consists of a solid-phase extraction (SPE) and subsequent HPLC with postcolumn derivatization of the amines with o-phthalaldehyde. The recovery rates of HA and tele-MHA during the SPE procedure were 82.8+/-3.4 and 86.0+/-1.7%, respectively. The detection limits for HA and tele-MHA were 8 and 12pg, respectively, with sufficient linearity up to 30pg. Using this newly developed system, we observed that the brain tele-MHA levels in H3 receptor knockout mice were significantly higher than those of wild-type mice by 2.1-fold. Furthermore, we also observed that the brain HA and tele-MHA levels in Zucker rats were significantly lower than those of lean rats by 76.6+/-5.3 and 77.8+/-5.0%, respectively. These observations coincided well with those of previous studies using radioimmunoassay or HPLC with precolumn OPA derivatization, confirming the utilization of the assay system.     

Stem cell factor has a suppressive activity to IgE-mediated chemotaxis of mast cells

Stem cell factor (SCF), which is well known as a cytokine capable of amplifying development and functions of mast cells, is mainly released from fibroblasts in the peripheral tissue. To investigate whether SCF controlled chemotactic migration of mast cells induced by IgE-specific Ag, murine bone marrow-derived cultured mast cells (BMCMC) and human cord blood-derived cultured mast cells (HuCMC) were preincubated with SCF. Although BMCMC and HuCMC sensitized with IgE directly moved toward specific Ag, preincubation for even 1 h with an optimal dose of SCF suppressed the IgE-mediated chemotactic movement. No or little inhibitory effect of SCF was detected in BMCMC derived from c-kit receptor-defect WBB6F1-W/Wv mice. In contrast, preincubation of BMCMC and HuCMC with SCF enhanced beta-hexosaminidase release and Ca2+ mobilization in response to Ag after sensitization with IgE. Using the real-time record of chemotactic migration, BMCMC preincubated with SCF manifested motionless without degranulation. These results suggest that locally produced SCF may have an inhibitory effect on chemotaxis of mast cells, contributing to their accumulation and enhancement of functions at the peripheral site in allergic and nonallergic conditions.     

Investigations on small molecule inhibitors targeting the histone H3K4 tri-methyllysine binding PHD-finger of JmjC histone demethylases

Plant homeodomain (PHD) containing proteins are important epigenetic regulators and are of interest as potential drug targets. Inspired by the amiodarone derivatives reported to inhibit the PHD finger 3 of KDM5A (KDM5A(PHD3)), a set of compounds were synthesised. Amiodarone and its derivatives were observed to weakly disrupt the interactions of a histone H3K4me3 peptide with KDM5A(PHD3). Selected amiodarone derivatives inhibited catalysis of KDM5A, but in a PHD-finger independent manner. Amiodarone derivatives also bind to H3K4me3-binding PHD-fingers from the KDM7 subfamily. Further work is required to develop potent and selective PHD finger inhibitors.     

Evolution of Mammalian Opn5 as a Specialized UV-absorbing Pigment by a Single Amino Acid Mutation

Opn5 is one of the recently identified opsin groups that is responsible for nonvisual photoreception in animals. We previously showed that a chicken homolog of mammalian Opn5 (Opn5m) is a G_i-coupled UV sensor having molecular properties typical of bistable pigments. Here we demonstrated that mammalian Opn5m evolved to be a more specialized photosensor by losing one of the characteristics of bistable pigments, direct binding of all-trans-retinal. We first confirmed that Opn5m proteins in zebrafish, Xenopus tropicalis, mouse, and human are also UV-sensitive pigments. Then we found that only mammalian Opn5m proteins lack the ability to directly bind all-trans-retinal. Mutational analysis showed that these characteristics were acquired by a single amino acid replacement at position 168. By comparing the expression patterns of Opn5m between mammals and chicken, we found that, like chicken Opn5m, mammalian Opn5m was localized in the ganglion cell layer and inner nuclear layer of the retina. However, the mouse and primate (common marmoset) opsins were distributed not in the posterior hypothalamus (including the region along the third ventricle) where chicken Opn5m is localized, but in the preoptic hypothalamus. Interestingly, RPE65, an essential enzyme for forming 11-cis-retinal in the visual cycle is expressed near the preoptic hypothalamus of the mouse and common marmoset brain but not near the region of the chicken brain where chicken Opn5m is expressed. Therefore, mammalian Opn5m may work exclusively as a short wavelength sensor in the brain as well as in the retina with the assistance of an 11-cis-retinal-supplying system.     

A Cluster of Thin Tubular Structures Mediates Transformation of the Endoplasmic Reticulum to Autophagic Isolation Membrane

Recent findings have suggested that the autophagic isolation membrane (IM) might originate from a domain of the endoplasmic reticulum (ER) called the omegasome. However, the morphological relationships between ER, omegasome, and IM remain unclear. In the present study, we found that hybrid structures composed of a double FYVE domain-containing protein 1 (DFCP1)-positive omegasome and the IM accumulated in Atg3-deficient mouse embryonic fibroblasts (MEFs). Moreover, correlative light and electron microscopy and immunoelectron microscopy revealed that green fluorescent protein (GFP)-tagged DFCP1 was localized on tubular or vesicular elements adjacent to the IM rims. Through detailed morphological analyses, including optimization of a fixation method and electron tomography, we observed a cluster of thin tubular structures between the IM edges and ER, part of which were continuous with IM and/or ER. The formation of these thin tubular clusters was observed in several cell lines and MEFs deficient for Atg5, Atg7, or Atg16L1 but not in FIP200-deficient cells, suggesting that they were relevant to the earlier events in autophagosome formation. Taken together, our findings indicate that these tubular profiles represent a part of the omegasome that links the ER with the IM.     

Studies on the catalytic domains of multiple JmjC oxygenases using peptide substrates

The JmjC-domain-containing 2-oxoglutarate-dependent oxygenases catalyze protein hydroxylation and N^ε-methyllysine demethylation via hydroxylation. A subgroup of this family, the JmjC lysine demethylases (JmjC KDMs) are involved in histone modifications at multiple sites. There are conflicting reports as to the substrate selectivity of some JmjC oxygenases with respect to KDM activities. In this study, a panel of modified histone H3 peptides was tested for demethylation against 15 human JmjC-domain-containing proteins. The results largely confirmed known N^ε-methyllysine substrates. However, the purified KDM4 catalytic domains showed greater substrate promiscuity than previously reported (i.e., KDM4A was observed to catalyze demethylation at H3K27 as well as H3K9/K36). Crystallographic analyses revealed that the N^ε-methyllysine of an H3K27me3 peptide binds similarly to N^ε-methyllysines of H3K9me3/H3K36me3 with KDM4A. A subgroup of JmjC proteins known to catalyze hydroxylation did not display demethylation activity. Overall, the results reveal that the catalytic domains of the KDM4 enzymes may be less selective than previously identified. They also draw a distinction between the N^ε-methyllysine demethylation and hydroxylation activities within the JmjC subfamily. These results will be of use to those working on functional studies of the JmjC enzymes.     

Recombinant human monoclonal antibodies to human cytomegalovirus glycoprotein B neutralize virus in a complement-dependent manner

Human antibodies specific for HCMV are currently considered as potential anti-HCMV therapeutic agents. In this study, we used a combinatorial human antibody library to isolate and characterize complete human monoclonal antibodies that effectively neutralize HCMV in a complement-dependent manner. One hundred and six clones were isolated in two independent screens using HCMV virions and recombinant glycoprotein B, gB654, as antigens. All of the clones recognized the same molecule gB and were classified into 14 groups based on the amino acid sequence of the V_H region. Seven representative clones from these 14 groups had a strong gB654 binding affinity by surface plasmon resonance (SPR). A pairwise binding competition analysis suggested that there were three groups based on differences in the gB recognition sites. Although Fab fragments of the seven groups showed strong affinity for gB, none of the Fab fragments neutralized HCMV infectivity in vitro. In contrast, complete human IgG₁ antibodies of at least three groups neutralized HCMV in a complement-dependent manner. These data suggest that potent therapeutic antibodies can be obtained from a human antibody library, including most of the functional antibodies that mediate humoral immunity to the selected pathogen.