Yoshiyuki Seyama

Direct evidence for the involvement of brain-derived neurotrophic factor in the development of a neuropathic pain-like state in mice

Thermal hyperalgesia and tactile allodynia induced by sciatic nerve ligation were completely suppressed by repeated intrathecal (i.t.) injection of a TrkB/Fc chimera protein, which sequesters endogenous brain‐derived neurotrophic factor (BDNF). In addition, BDNF heterozygous (+/–) knockout mice exhibited a significant suppression of nerve ligation‐induced thermal hyperalgesia and tactile allodynia compared with wild‐type mice. After nerve ligation, BDNF‐like immunoreactivity on the superficial laminae of the ipsilateral side of the spinal dorsal horn was clearly increased compared with that of the contralateral side. It should be noted that a single i.t. injection of BDNF produced a long‐lasting thermal hyperalgesia and tactile allodynia in normal mice, and these responses were abolished by i.t. pre‐treatment with either a Trk‐dependent tyrosine kinase inhibitor K‐252a or a selective protein kinase C (PKC) inhibitor Ro‐32‐0432. Supporting these findings, we demonstrated here for the first time that the increase in intracellular Ca2+ concentration by application of BDNF in cultured mouse spinal neurons was abolished by pre‐treatment with either K‐252a or Ro‐32‐0432. Taken together, these findings suggest that the binding of spinally released BDNF to TrkB by nerve ligation may activate PKC within the spinal cord, resulting in the development of a neuropathic pain‐like state in mice.

Role of δ-opioid receptor function in neurogenesis and neuroprotection

The present study was undertaken to evaluate the implication of δ‐opioid receptor function in neurogenesis and neuroprotection. We found that the stimulation of δ‐opioid receptors by the selective δ‐opioid receptor agonist SNC80 [(+)‐4‐[(αR)‐α‐((2S,5R)‐4‐allyl‐2,5‐dimethyl‐1‐piperazinyl)‐3‐methoxybenzyl]‐N,N‐diethylbenzamide] (10 nm) promoted neural differentiation from multipotent neural stem cells obtained from embryonic C3H mouse forebrains. In contrast, either a selective µ‐opioid receptor agonist, [d‐Ala2, N‐Me‐Phe4, Gly5‐ol]‐enkephalin (DAMGO), or a specific κ‐opioid receptor agonist, (–)‐trans‐(1S,2S)‐U‐50488 hydrochloride (U50,488H), had no such effect. In addition to neural differentiation, the increase in cleaved caspase 3‐like immunoreactivity induced by H2O2 (3 µm) was suppressed by treatment with SNC80 in cortical neuron/glia co‐cultures. These effects of SNC80 were abolished by a Trk‐dependent tyrosine kinase inhibitor: (8R*,9S*,11S*)‐(–)‐9‐hydroxy‐9‐methoxycarbonyl‐8‐methyl‐2,3,9,10‐tetrahydro‐8,11‐epoxy‐1H,8H,11H‐2,7b,11a‐triazadibenzo(a,g)cycloocta(cde)trinden‐1‐one (K‐252a). The SNC80‐induced neural differentiation was also inhibited by treatment with the protein kinase C (PKC) inhibitor, phosphatidylinositol 3‐kinase (PI3K) inhibitor, mitogen‐activated protein kinase kinase (MEK) inhibitor or Ca2+/calmodulin‐dependent protein kinase II (CaMKII) inhibitor. These findings raise the possibility that δ‐opioid receptors play a crucial role in neurogenesis and neuroprotection, mainly through the activation of Trk‐dependent tyrosine kinase, which could be linked to PI3K, PKC, CaMKII and MEK.

Chronic pain-induced emotional dysfunction is associated with astrogliosis due to cortical delta-opioid receptor dysfunction.

It has been widely recognized that chronic pain could cause physiological changes at supraspinal levels. The δ‐opioidergic system is involved in antinociception, emotionality, immune response and neuron‐glia communication. In this study, we show that mice with chronic pain exhibit anxiety‐like behavior and an increase of astrocytes in the cingulate cortex due to the dysfunction of cortical δ‐opioid receptor systems. Using neural stem cells cultured from the mouse embryonic forebrain, astrocyte differentiation was clearly observed following long‐term exposure to the selective δ‐opioid receptor antagonist, naltrindole. We also found that micro‐injection of either activated astrocyte or astrocyte‐conditioned medium into the cingulate cortex of mice aggravated the expression of anxiety‐like behavior. Our results indicate that the chronic pain process promotes astrogliosis in the cingulate cortex through the dysfunction of cortical δ‐opioid receptors. This phenomenon may lead to emotional disorders including aggravated anxiety under chronic pain‐like state.

Stimulation of cell proliferation and autoregulation of elastin expression by elastin peptide VPGVG in cultured chick vascular smooth muscle cells

Synthetic elastin peptides, VPGVG or its polymer (VPGVG) n , enhanced the proliferation of smooth muscle cells 1.5‐fold during 48 h treatment at the concetrations over 10−6 M or 1.0 μg/ml, respectively. Monomeric and polymeric VPGVG sequences reduced elastin synthesis and its mRNA level to one‐third and one‐half of control respectively under the conditions in which the proliferation of cells were enhanced, but did not change collagen synthesis as measured by bacterial collagenase digestion. The elastin‐specific autoregulation by elastin fragments may reflect the feedback regulation of elastin expression which may play an essential role in elastin metabolism under the normal and diseased conditions.

Characterization of the Molecular Interaction between Tropoelastin and DANCE/Fibulin-5

Fibulin-5 is believed to play an important role in the elastic fiber formation. The present experiments were carried out to characterize the molecular interaction between fibulin-5 and tropoelastin. Our data showed that the divalent cations of Ca(2+), Ba(2+) and Mg(2+) significantly enhanced the binding of fibulin-5 to tropoelastin. In addition, N-linked glycosylation of fibulin-5 does not require for the binding to tropoelastin. To address the fibulin-5 binding site on tropoelastin constructs containing, exons 2-15 and exons 16-36, of tropoelastin were used. Fibulin-5 binding was significantly reduced to either fragment and also to a mixture of the two fragments. These results suggested that the whole molecule of tropoelastin was required for the interaction with fibulin-5. In co-immunoprecipitation experiments, tropoelastin binding to fibulin-5 was enhanced by an increase of temperature and sodium chloride concentration, conditions that enhance the coacervation of tropoelastin. The binding of tropoelastin fragments to fibulin-5 was directly proportional to their propensity to coacervate. Furthermore, the addition of fibulin-5 to tropoelastin facilitated coacervation. Taken together, the present study shows that fibulin-5 enhances elastic fiber formation in part by improving the self-association properties of tropoelastin.

Domains 16 and 17 of tropoelastin in elastic fibre formation

Naturally occurring mutations are useful in identifying domains that are important for protein function. We studied a mutation in the elastin gene, 800-3G>C, a common disease allele for SVAS (supravalvular aortic stenosis). We showed in primary skin fibroblasts from two different SVAS families that this mutation causes skipping of exons 16-17 and results in a stable mRNA. Tropoelastin lacking domains 16-17 (Delta16-17) was synthesized efficiently and secreted by transfected retinal pigment epithelium cells, but showed the deficient deposition into the extracellular matrix compared with normal as demonstrated by immunofluorescent staining and desmosine assays. Solid-phase binding assays indicated normal molecular interaction of Delta16-17 with fibrillin-1 and fibulin-5. However, self-association of Delta16-17 was diminished as shown by an elevated coacervation temperature. Moreover, negative staining electron microscopy confirmed that Delta16-17 was deficient in forming fibrillar polymers. Domain 16 has high homology with domain 30, which can form a beta-sheet structure facilitating fibre formation. Taken together, we conclude that domains 16-17 are important for self-association of tropoelastin and elastic fibre formation. This study is the first to discover that domains of elastin play an essential role in elastic fibre formation by facilitating homotypic interactions.

DANCE/fibulin-5 promotes elastic fiber formation in a tropoelastin isoform-dependent manner

OBJECTIVE To investigate a function of fibulin-5 in the elastic fiber formation, we studied the molecular interactions among elastin, fibrillin-1, and fibulin-5 in the extracellular space and the maturation of tropoelastin using retinal pigment epithelial cells (ARPE-19). DESIGN AND METHODS Bacterial recombinant tropoelastin (rTE) was added to ARPE-19 cells overexpressing V5-tagged fibulin-5 (ARPE-Fibulin-5). These elastic fibers were evaluated by immunofluorescence staining, the quantitative analysis of cross-linked amino acids, and semi-quantitative analysis of matrix-associated tropoelastin. RESULTS Immunoprecipitation assays revealed that fibulin-5 is able to separately interact with tropoelastin or fibrillin-1 in the culture medium. Moreover, immunofluorescent staining showed that elastin, fibrillin-1, and fibulin-5 co-localize in the extracellular matrix. Desmosine levels were significantly increased in ARPE-Fibulin-5 relative to untransfected cells in spite of equal deposition of tropoelastin by enzyme-linked immunosorbent assay. The addition of a tropoelastin isoform, which lacked the peptide encoded by exon 26A (Delta26A) and could bind to fibulin-5 strongly, led to a larger increase in cross-linking amino acids compared to tropoelastin containing the exon 26A peptide sequence. CONCLUSION These data provide new insights into the initial steps of elastic fiber assembly and identify fibulin-5 and tropoelastin isoforms as potential targets for the regeneration of elastic fibers in vivo.

Separation of stereoisomers of several furan derivatives by capillary gas chromatography-mass spectrometry, supercritical fluid chromatography, and liquid chromatography using chiral stationary phases.

The direct separation of several stereoisomers (enantiomers and geometrical isomers) of furan derivatives, important intermediates for the synthesis of physiologically active natural products, was achieved using capillary gas chromatography/mass spectrometry with a per-O-methyl-beta-cyclodextrin, supercritical fluid chromatography and high-performance liquid chromatography with a tris(3,5-dimethylphenylcarbamate) of cellulose or amylose for the chiral stational phases, respectively. The temperature dependence of the peak resolution (Rs) and the retention factor (k) over the range of 110-130 degrees was studied using crotyl furfuryl ether in gas chromatography. Successive increases in the Rs value and of the difference between the k value of the E-isomer and the k value of the Z-isomer were observed when the gradient temperature was decreased. The per-O-methyl-beta-cyclodextrin column was suitable for use with volatile furan ethers whose molecular masses are between 150 and 180. In conclusion, the separation of thermally unstable furan derivatives was accomplished using supercritical fluid chromatography and high-performance liquid chromatography.

Phorbol Ester-dependent Phosphorylation Regulates the Association of p57/Coronin-1 with the Actin Cytoskeleton

The p57/coronin-1 protein is a member of the coronin family of actin-binding proteins, which are characterized by the presence of WD (tryptophan/aspartic acid) repeats and a coiled-coil motif in the molecule. It is selectively expressed in immune cells and has been suggested to play crucial roles in leukocyte functions, including cell migration and phagocytosis. In this study we examined the effects of p57/coronin-1 phosphorylation on the association of the protein with actin. Treatment of HL60 human leukemic cells or p57/coronin-1-transfected HEK293 cells with phorbol 12-myristate 13-acetate (PMA) reduced the association of p57/coronin-1 with the actin cytoskeleton, as indicated by cell fractionation experiments and by fluorescence microscopic observation. Two-dimensional gel electrophoresis of HL60 cell lysate revealed that p57/coronin-1 was phosphorylated upon PMA stimulation of the cells, giving two major and two minor spots of phosphorylated forms, each with distinct isoelectric points. The p57/coronin-1 molecules associated with the cytoskeleton in PMA-treated HL60 cells were phosphorylated at lower levels than those recovered in the cytosolic fraction. In addition, p57/coronin-1 co-sedimented with F-actin polymerized in vitro had lower phosphorylation levels than the molecules remaining in the supernatant. By affinity chromatographic analysis using anti-p57/coronin-1 antibody-conjugated Sepharose, p57/coronin-1 derived from PMA-treated HL60 cells showed lower affinity for actin than that from untreated cells. Finally, recovery of p57/coronin-1 in the actin cytoskeleton-rich fraction from neutrophil-like differentiated HL60 cells decreased during phagocytosis, concomitant with enhanced phosphorylation of p57/coronin-1. These results strongly suggest that the phosphorylation of p57/coronin-1 down-regulates its association with actin and modulates the reorganization of actin-containing cytoskeleton.

Comparative Effects of Vitamin K2 and Vitamin E on Experimental Arteriosclerosis

The comparative effects of vitamin K2 and vitamin E on aortic calcium (Ca) and inorganic phosphorus (P) levels in the aorta and the elastin fraction (fr.) were investigated in male rats after experimental arteriosclerosis was induced by vitamin D2 with atherogenic diet. Both vitamin K2 (100 mg/kg b.w.) and vitamin E (40 mg/kg b.w.) inhibited the increase of Ca and P in the aorta and the elastin fr. from the arteriosclerotic rats. Vitamin K2 (50 mg/kg b.w.) also suppressed the deposition of Ca and P in the aorta, but there was no change due to vitamin K3 or geranylgeraniol (side chain of vitamin K2) administration. Both vitamin K2 and vitamin E showed lipid radical scavenging activity in the in vitro experiment. However, neither vitamin K3 nor geranylgeraniol exhibited anti-arteriosclerotic or radical scavenging activity under the above experimental conditions. It is suggested that vitamin K2 and vitamin E promoted an antiarteriosclerotic effect by radical scavenging activity. These actions of vitamin K2 are required in the structure of 2-methylnaphtoquinone and its side chain (geranylgeraniol).