Calcitonin Gene-Related Peptide Regulates Type IV Hypersensitivity through Dendritic Cell Functions

Dendritic cells (DCs) play essential roles in both innate and adaptive immune responses. In addition, mutual regulation of the nervous system and immune system is well studied. One of neuropeptides, calcitonin gene-related peptide (CGRP), is a potent regulator in immune responses; in particular, it has anti-inflammatory effects in innate immunity. For instance, a deficiency of the CGRP receptor component RAMP 1 (receptor activity-modifying protein 1) results in higher cytokine production in response to LPS (lipopolysaccharide). On the other hand, how CGRP affects DCs in adaptive immunity is largely unknown. In this study, we show that CGRP suppressed Th1 cell differentiation via inhibition of IL-12 production in DCs using an in vitro co-culture system and an in vivo ovalbumin-induced delayed-type hypersensitivity (DTH) model. CGRP also down-regulated the expressions of chemokine receptor CCR2 and its ligands CCL2 and CCL12 in DCs. Intriguingly, the frequency of migrating CCR2⁺ DCs in draining lymph nodes of RAMP1-deficient mice was higher after DTH immunization. Moreover, these CCR2⁺ DCs highly expressed IL-12 and CD80, resulting in more effective induction of Th1 differentiation compared with CCR2⁻ DCs. These results indicate that CGRP regulates Th1 type reactions by regulating expression of cytokines, chemokines, and chemokine receptors in DCs.     

A genetically encoded Förster resonance energy transfer biosensor for two-photon excitation microscopy

Pippi (phosphatidyl inositol phosphate indicator) is a biosensor based on the principle of FRET (Förster resonance energy transfer), which consists of a pair of fluorescent proteins, CFP (cyan fluorescent protein) and YFP (yellow fluorescent protein), the PH domain sandwiched between them, and K-Ras C-terminal sequence for plasma membrane localization. Due to marked cross-excitation of YFP with the conditions used to excite CFP, initial FRET images obtained by TPE (two-photon excitation) microscopy suffered from low signal-to-noise ratio, hampering the observation of lipids in three-dimensional structures. To solve this problem, YFP and CFP in the original Pippi-PI(3,4)P₂ was replaced by sREACh (super resonance energy accepting chromoprotein) and mTFP1 (monomeric teal fluorescent protein), respectively. The biosensor was also fused with an internal control protein, mKeima, where Keima/mTFP1 indicates the FRET efficiency, and indeed epidermal growth factor stimulation increased Keima/mTFP1 in HeLa cells. This biosensor successfully showed PI(3,4)P₂ accumulation to the lateral membrane in the MDCK cyst cultured in a three-dimensional environment. Furthermore, other FRET-based biosensors for PIP₃ distribution and for tyrosine kinase activity were developed based on this method, suggesting its broad application for visualizing signal transduction events with TPE microscopy.     

Autocrine activation of EGF receptor promotes oscillation of glutamate-induced calcium increase in astrocytes cultured in rat cerebral cortex

We previously reported that astrocytes cultured for more than 2 days in a defined medium containing epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) showed calcium oscillation in response to glutamate, whereas the response pattern was transient in the absence of the exogenous growth factors. In the present study, we found that astrocytes showed glutamate-induced calcium oscillation, even in growth factor-free medium, if the cells had been cultured for more than 5 days. The calcium oscillation promoted by the prolonged culture period was suppressed by an inhibitor of EGF receptor tyrosine kinase, but not by a neutralizing antibody to bFGF, indicating that the accumulation of an autocrine factor that activates the EGF receptor leads to calcium oscillation. Astrocytes in our culture system expressed EGF, transforming growth factor alpha (TGFalpha), bFGF and acidic fibroblast growth factor (aFGF). Exogenous aFGF, which induced astrocyte immediate early gene expression to the same extent as EGF or bFGF, did not affect calcium oscillation. Exogenous EGF and bFGF promoted astrocyte hypertrophic morphology and proliferation, as well as calcium oscillation. In contrast, these properties did not accompany calcium oscillation induced by the prolonged culture period. These results suggest that astrocytes possess the ability to promote their own calcium oscillation, which is independent of hypertrophic changes to reactive astrocytes.     

The N-terminus of B96Bom, a Bombyx mori G-protein-coupled receptor, is N-myristoylated and translocated across the membrane

In eukaryotic cellular proteins, protein N-myristoylation has been recognized as a protein modification that occurs mainly on cytoplasmic or nucleoplasmic proteins. In this study, to search for a eukaryotic N-myristoylated transmembrane protein, the susceptibility of the N-terminus of several G-protein-coupled receptors (GPCRs) to protein N-myristoylation was evaluated by in vitro and in vivo metabolic labeling. It was found that the N-terminal 10 residues of B96Bom, a Bombyx mori GPCR, efficiently directed the protein N-myristoylation. Analysis of a tumor necrosis factor (TNF) fusion protein with the N-terminal 90 residues of B96Bom at its N-terminus revealed that (a) transmembrane domain 1 of B96Bom functioned as a type I signal anchor sequence, (b) the N-myristoylated N-terminal domain (58 residues) was translocated across the membrane, and (c) two N-glycosylation motifs located in this domain were efficiently N-glycosylated. In addition, when Ala4 in the N-myristoylation motif of B96Bom90-TNF, Met-Gly-Gln-Ala-Ala-Thr(1-6), was replaced with Asn to generate a new N-glycosylation motif, Asn-Ala-Thr(4-6), efficient N-glycosylation was observed on this newly introduced N-glycosylation site in the expressed protein. These results indicate that the N-myristoylated N-terminus of B96Bom is translocated across the membrane and exposed to the extracellular surface. To our knowledge, this is the first report showing that a eukaryotic transmembrane protein can be N-myristoylated and that the N-myristoylated N-terminus of the protein can be translocated across the membrane.     

Characterization of linear-oblong pyrenoids with cp-DNA along their sides in Nitzschia sigmoidea (Bacillariophyceae)

In the past 10 years Nitzschia sigmoidea (Nitzsch) W. Sm. has begun to occur in Japanese rivers in various areas. It is a common diatom in Europe but was previously absent in Japan. Each chloroplast of N. sigmoidea contains many unusual linear‐oblong structures. The internal structure of the chloroplast in this species was observed using epifluorescence and electron microscopy with immunolocalization techniques. The linear‐oblong structures in the chloroplasts could hardly be observed by conventional light microscopy of living cells, but were obvious in cells stained with propionocarmine. Transmission electron microscopy showed that the cross sections of this structure were lanceolate to fusiform with penetration by a single thylakoid. In cells stained with DAPI, chloroplast DNA was detected along both sides of the linear‐oblong structures, and DNA fibrils were detected by electron microscopy. Immunofluorescence microscopy of sectioned cells and also immunoelectron microscopy revealed specific localization of Rubisco between these DNA‐containing areas, which divided at the same time as the chloroplast. Our observations confirmed that the linear‐oblong structures are pyrenoids. The diversity of localization patterns of chloroplast DNA in diatoms is discussed.     

Thrombin-induced rapid geranylgeranylation of RhoA as an essential process for RhoA activation in endothelial cells

RhoA plays a critical signaling role in thrombin-induced endothelial dysfunction. The possible thrombin regulation of geranylgeranylation, a lipid modification, of unprocessed RhoA and the significance of the geranylgeranylation in RhoA activation in endothelial cells (ECs) are not well understood. The amounts of the unprocessed and geranylgeranylated forms of RhoA in non-stimulated cultured human aortic ECs were 31 +/- 8 and 69 +/- 8% total cellular RhoA, respectively (n = 6, p < 0.0001), as determined by the Triton X-114 partition method. Thrombin-induced rapid conversion of most of the unprocessed RhoA into the geranylgeranylated form within 1 min through stimulating geranylgeranyltransferase I (GGTase I) activity. Thrombin-induced rapid geranylgeranylation was inhibited by acute short term (3 min) pretreatment with atorvastatin as well as by an inhibitor of GGTase I (GGTI-286). Thrombin also rapidly stimulated GTP loading of RhoA, which was blocked by acute pretreatment with either atorvastatin or GGTI-286. These observations indicate the dependence of thrombin stimulation of RhoA on the rapid geranylgeranylation of unprocessed RhoA. Importantly, the addition of geranylgeranylpyrophosphate to ECs pretreated with atorvastatin quickly reversed the atorvastatin inhibition of thrombin stimulation of RhoA. These results suggest that geranylgeranylation of unprocessed RhoA may limit thrombin-induced full activation of RhoA in ECs. Cytoskeleton analysis demonstrated that atorvastatin and GGTI-286 inhibited thrombin-induced stress fiber formation. We provide the evidence that, in thrombin-stimulated ECs, the unprocessed form of RhoA is rapidly geranylgeranylated to become the mature form, which then is converted into GTP-bound active RhoA.     

Role of LOX‐1 in monocyte adhesion‐triggered redox,Akt/eNOS and Ca2+ signaling pathways in endothelial cells

This study was conducted to examine the role of lectin‐like oxidized low‐density lipoprotein receptor‐1 (LOX‐1) in monocyte adhesion‐induced redox‐sensitive, Akt/eNOS and Ca²⁺ signaling pathways in endothelial cells (ECs). LOX‐1 was blocked by an antibody‐neutralizing LOX‐1 TS92 or small interfering RNA. In cultured human aortic ECs, monocyte adhesion activated Rac1 and p47^phox, and increased NADPH oxidase activity and reactive oxygen species (ROS) generation within 30 min and NF‐κB phosphorylation within 1 h, resulting in redox‐sensitive gene expression. Akt and eNOS phosphorylation was induced 15 min after adding monocytes and returned to control level after 30 min, whereas NO production was not altered by monocyte adhesion. Blockade of LOX‐1 blunted the monocyte adhesion‐triggered redox‐sensitive signaling pathway and Akt/eNOS phosphorylation in ECs. Both endothelial intracellular Ca²⁺ mobilization and Ca²⁺ influx caused by monocyte attachment were markedly attenuated by pretreatment of ECs with TS92. This suggests that LOX‐1 is involved in redox‐sensitive, Akt/eNOS and Ca²⁺ signaling pathways in monocyte adhesion to ECs independent of oxidized low‐density lipoprotein (ox‐LDL). Furthermore, blockade of Ca²⁺ inhibited monocyte adhesion‐triggered Rac1 and p47^phox activation and ROS generation in ECs, whereas Ca²⁺ signaling was suppressed by blockade of NADPH oxidase and ROS generation. Finally, TS92 blocked the monocyte adhesion to ECs stimulated with or without tumor necrosis factor‐α or ox‐LDL. We provide evidence that LOX‐1 plays a role in redox‐sensitive, Akt/eNOS and Ca²⁺ signaling pathways in monocyte adhesion to ECs independent of the ox‐LDL–LOX‐1 axis. J. Cell. Physiol. 220: 706–715, 2009. © 2009 Wiley‐Liss, Inc.     

High-resolution image-based simulation reveals membrane strain concentration on osteocyte processes caused by tethering elements

Osteocytes are vital for regulating bone remodeling by sensing the flow-induced mechanical stimuli applied to their cell processes. In this mechanosensing mechanism, tethering elements (TEs) connecting the osteocyte process with the canalicular wall potentially amplify the strain on the osteocyte processes. The ultrastructure of the osteocyte processes and canaliculi can be visualized at a nanometer scale using high-resolution imaging via ultra-high voltage electron microscopy (UHVEM). Moreover, the irregular shapes of the osteocyte processes and the canaliculi, including the TEs in the canalicular space, should considerably influence the mechanical stimuli applied to the osteocytes. This study aims to characterize the roles of the ultrastructure of osteocyte processes and canaliculi in the mechanism of osteocyte mechanosensing. Thus, we constructed a high-resolution image-based model of an osteocyte process and a canaliculus using UHVEM tomography and investigated the distribution and magnitude of flow-induced local strain on the osteocyte process by performing fluid–structure interaction simulation. The analysis results reveal that local strain concentration in the osteocyte process was induced by a small number of TEs with high tension, which were inclined depending on the irregular shapes of osteocyte processes and canaliculi. Therefore, this study could provide meaningful insights into the effect of ultrastructure of osteocyte processes and canaliculi on the osteocyte mechanosensing mechanism.

     

Chromones from Harrisonia perforata

Five new chromones, perforatins C-G, together with 10 known compounds were isolated from the wood of Harrisonia perforata.