GCN2 is the alpha-subunit of the only translation initiation factor (eIF2alpha) kinase that appears in all eukaryotes. Its function requires an interaction with GCN1 via the domain at its N-terminus, which is termed the RWD domain after three major RWD-containing proteins: RING finger-containing proteins, WD-repeat-containing proteins, and yeast DEAD (DEXD)-like helicases. In this study, we determined the solution structure of the mouse GCN2 RWD domain using NMR spectroscopy. The structure forms an alpha + beta sandwich fold consisting of two layers: a four-stranded antiparallel beta-sheet, and three side-by-side alpha-helices, with an alphabetabetabetabetaalphaalpha topology. A characteristic YPXXXP motif, which always occurs in RWD domains, forms a stable loop including three consecutive beta-turns that overlap with each other by two residues (triple beta-turn). As putative binding sites with GCN1, a structure-based alignment allowed the identification of several surface residues in alpha-helix 3 that are characteristic of the GCN2 RWD domains. Despite the apparent absence of sequence similarity, the RWD structure significantly resembles that of ubiquitin-conjugating enzymes (E2s), with most of the structural differences in the region connecting beta-strand 4 and alpha-helix 3. The structural architecture, including the triple beta-turn, is fundamentally common among various RWD domains and E2s, but most of the surface residues on the structure vary. Thus, it appears that the RWD domain is a novel structural domain for protein-binding that plays specific roles in individual RWD-containing proteins.

Amino Acid Sequence↗

PubMed2004

Low dopamine d(2) receptor binding in subregions of the thalamus in schizophrenia.

OBJECTIVE: Several structural and functional brain imaging studies have pointed to a disturbance of thalamic subnuclei in patients with schizophrenia. The dopamine hypothesis of schizophrenia has, however, not been thoroughly examined in terms of this complex structure, which has connections with most brain regions of central interest in schizophrenia research. The aim of the present study was to examine dopamine D(2) receptor binding in subregions of the thalamus in patients with schizophrenia. METHOD: The authors used positron emission tomography and the radioligand [(11)C]FLB457 to examine dopamine D(2) receptor binding in thalamic subregions of 10 drug-naive patients with schizophrenia. Binding potential was calculated by the reference tissue method and used as an index for dopamine D(2) receptor binding. Comparisons were made with 19 healthy subjects. Subregions of interest were defined on individual magnetic resonance images using a percentage-based operational approach. Clinical symptoms were rated by using the Brief Psychiatric Rating Scale (BPRS). RESULTS: The [(11)C]FLB457 binding potential was lower in the central medial and posterior subregions of the thalamus in patients with schizophrenia. At a functional level, there was a significant negative correlation between binding potential and BPRS positive symptom scores. CONCLUSIONS: The subregions with low D(2) receptor binding comprise primarily the dorsomedial nucleus and pulvinar, two important components in circuitries previously suggested in the pathophysiology of schizophrenia. Aberrant dopaminergic neurotransmission in thalamic subregions might be a mechanism underlying positive symptoms in schizophrenia.

Adult↗

PubMed2004

Antioxidant and immuno-enhancing effects of Echinacea purpurea.

We studied the protective effects of Echinacea purpurea against radiation by evaluating changes in the peripheral blood cell count and peripheral blood antioxidant activity. E. purpurea administration had a suppressive effect on radiation-induced leukopenia, especially on lymphocytes and monocytes, and resulted in a faster recovery of blood cell counts. Mouse peripheral blood antioxidant activity was increased by E. purpurea, and a relationship between the suppressive effect on radiation-induced leukopenia and the antioxidant effect was suggested. Furthermore, we reviewed the evidence of augmentation of found in this study humoral immunity. The effects of immune activation by E. purpurea were investigated by measuring total immunoglobulin (IgG, IgM). The radioprotective effects of immune activation by E. purpurea were investigated by measuring T lymphocyte subsets in the peripheral blood of mice following whole-body irradiation. E. purpurea activates macrophages to stimulate IFN-gamma production in association with the secondary activation of T lymphocytes, resulting in a decrease in IgG and IgM production. Cytokines released from macrophages in mouse peripheral blood after E. purpurea administration activated helper T cells to proliferate. In addition, it is reported that activated macrophages in association with the secondary T lymphocyte activation increases IFN-gamma production and stimulates proliferation of cytotoxic T cells and suppressor T cells. We think that CD 4 and CD 8 subsets were more immunologically enhanced by E. purpurea than helper T cells and suppressor T cell these results reflect activation. In addition, we think that these results reflect cell-mediated immune responses.

Adjuvants, Immunologic↗

PubMed2004

Suppressive effect of Kanzo-bushi-to, a Kampo medicine, on collagen-induced arthritis.

Kanzo-bushi-to (KBT) is a traditional Japanese herbal medicine (Kampo medicine), which is used in Japan to treat rheumatoid arthritis. In the present study, we investigated the suppressive effect of KBT on collagen-induced arthritis (CIA) and further studied the underlying mechanism. CIA was induced in male DBA/1J mice by immunization with bovine type II collagen, followed by a booster injection 21 d later. KBT was given at a dose of 430 mg/kg/d from three days before the first immunization to the end of the experiment. KBT suppressed CIA development effectively and further protected focal bone erosion and bone destruction as evidenced by the reduced histological score. Histochemical examination revealed that KBT decreased TRAP-positive cells at the synovium-bone interface and at the sites of focal bone erosion, coincident with the findings that RANKL/OPG mRNA ratio was significantly reduced by KBT treatment. KBT also decreased mRNA levels of M-CSF and iNOS in joints and of iNOS in peritoneal macrophages. In conclusion, KBT prevented osteoclast generation by decreasing RANKL/OPG ratio and M-CSF mRNA levels, resulting in reduction in bone erosion and destruction. In addition, KBT has anti-inflammatory effect such as the suppression of iNOS expression in peritoneal macrophages and joints of CIA mice. These finding suggests that KBT is a potential new therapeutic agent for the treatment of RA.

Animals↗

PubMed2004

Suppressive effect of Dai-bofu-to on collagen-induced arthritis.

Dai-bofu-to (DBT) is a traditional Japanese herbal medicine (Kampo medicine) used for the treatment of rheumatoid arthritis (RA). In the present study, to establish the usefulness of DBT, we examined the effect of DBT on collagen-induced arthritis (CIA). DBT (1.72 g/kg/d) significantly reduced the severity of arthritis throughout the experiment and significantly delayed the onset of arthritis. The induction of CIA decreased T cells and increased B cells in popliteal lymph nodes close to the affected joints, while the treatment of CIA with DBT counteracted the changes in T and B cells. In pX transgenic mice as a spontaneously developed arthritis model, a decrease in T cells and increase in B cells in popliteal lymph nodes were observed, as compared to BALB/c mice, the littermates of pX transgenic mice. In contrast, DBT returned the cell number of T and B cells to the level of BALB/c mice. As osteoclastogenesis is regulated by some T cell cytokines and osteotropic factors, we examined the effect of DBT on the receptor activator of NF-kappa B (RANK), RANK ligand (RANKL), osteoprotegerin (OPG) and M-CSF mRNAs, which were induced by arthritis induction. Although DBT had no effect on RNAK or RANKL mRNA levels, DBT stimulated an increase in OPG mRNA levels and suppressed an increase in M-CSF mRNA level. These results suggest that DBT may possess an anti-osteoclastogenetic effect, which is brought by reducing the ratio of RANKL/OPG and by decreasing M-CSF mRNA levels. In conclusion, immunomodulatory and anti-osteoclastogenetic effects might be involved in the suppression of arthritis by DBT.

Animals↗

PubMed2004

[Intravitreal injection of triamcinolone acetonide for cystoid macular edema resistant to vitreous surgery].

PURPOSE: To report the efficacy of intravitreal injection of triamcinolone acetonide(TA) for cystoid macular edema remaining after vitreous surgery. METHODS: Eight eyes of 7 patients aged from 32 to 84 years old were studied. The original diseases that caused macular edema were central retinal vein occlusion in 2 eyes, branch retinal vein occlusion in one eye, Irvine-Gass syndrome in 2 eyes, and diabetic retinopathy in 3 eyes. 4 mg of TA was injected intravitreally and visual acuity and foveal thickness measured by optical coherence tomography(OCT) were evaluated in before and after the surgery. RESULTS: Macular edema resolved rapidly after injection of TA in all cases and cysts were extinguished or diminished. The foveal thickness in OCT was reduced significantly from preoperative 495 +/- 116 (mean +/- standard deviation) microns to 267 +/- 117 microns after one week, and 246 +/- 81 microns after one month. The effect persisted for three months. Visual improvement of more than two Snellen lines was seen in 4 eyes. No side effects were observed except a temporary increase of intraocular pressure in one eye. CONCLUSION: Intravitreal injection of TA is effective in a short-term for cystoid macular edema remaining after vitrectomy.

Adult↗

PubMed2004

[Genetic diagnosis of retinoblastoma by a combination of fluorescence in situ hybridization and restriction fragment length polymorphism].

PURPOSE: It is important to exclude germ line mutation in cases of unilateral retinoblastoma(RB) to estimate hereditary or possible secondary cancer. We investigated whether genetic diagnosis is feasible in a health check screening program. METHODS: Five patients with RB had surgery for enucleation in Keio University Hospital. Tumor cells from enucleated eyes and lymphocytes representing systemic cells were collected and analyzed genetically by fluorescence in situ hybridization(FISH) and restriction fragment length polymorphism (RFLP). RESULTS: One out of three unilateral RB cases could be diagnosed as non-hereditary by the finding of no copies of the RB gene in the tumor cells using the FISH method and no signal in the RFLP method. A decrease of signal in tumor cells to less than 50% in the RFLP method was observed in another case of unilateral RB that seemed to be non-hereditary, but the case ultimately could not be diagnosed as non-hereditary because polycopies were found in the FISH method. No abnormality in tumor cells could be found in another unilateral case or in systemic cells of two bilateral cases. CONCLUSION: A combination of FISH and RFLP methods can be used to diagnose some cases of RB as non-hereditary.

Child, Preschool↗

PubMed2003

New approaches to study the development of morphine tolerance and dependence.

Morphine is now believed not to cause tolerance and dependence when it is appropriately used in clinic. However, in terminal cancer pain, patients' analgesic tolerance to morphine is developed due to the use of high doses of morphine for complete blockade of pain. At higher doses, morphine has more opportunity to show serious side effects, which worsens quality of life (QOL), and leads to the use of potent analgesic adjuvants to reduce the morphine dosage. Here we attempt to summarize recent studies of the molecular basis of morphine tolerance and dependence, and to discuss whether these mechanisms could provide new molecular targets as analgesic adjuvants. They include protein kinase C inhibitor, opioid agonist with low RAVE value, and antagonists of antiopioid receptors (GluRepsilon1 or nociceptin/OFQ receptor). In addition, we demonstrate new approaches to find further candidates of such molecular targets. These approaches include the visualization of neuronal networks in the downstream of opioid neurons by use of the WGA transgene technique and the single cell dissection technique to get new genes involved in plasticity during morphine tolerance and dependence.

Analgesics, Opioid↗

PubMed2003

Switching of bradykinin-mediated nociception following partial sciatic nerve injury in mice.

Bradykinin (BK) is well known as a potent mediator of pain and hyperalgesia. Using a highly sensitive nociception test, we found that intraplantar (i.pl.) injection of BK produced nociceptive hyper-responses in partial sciatic nerve-injured mice, compared with the control sham-operated animals. By use of selective agonists and antagonists, we revealed that BK nociception in sham-operated mice was mediated through B2 receptor, whereas that in injured mice was mediated through B1 receptor. When we examined the activation of extracellular signal-regulated protein kinase (ERK) in dorsal root ganglion (DRG) neurons upon i.pl. injection of BK, phosphorylated ERK was mainly observed in unmyelinated neurons in sham-operated mice, and in case of nerve-injured mice, ERK was mainly activated in myelinated neurons and satellite cells. The B1 receptor agonist, [Lys-des-Arg(9)]-BK also produced nociceptive response and activated ERK only in nerve-injured mice. BK or B1 agonist-induced activation of ERK in DRG neurons of nerve-injured mice was completely blocked by pretreatment with antisense oligodeoxynucleotide (AS-ODN) for B1 receptor. We found that in sham-operated mice mainly B2 receptors were expressed in unmyelinated DRG neurons with a very little presence of B1 receptor. After nerve injury, B2 receptor expression drastically decreased, whereas B1 receptors were newly expressed mainly in myelinated DRG neurons and satellite cells. Finally, BK nociception in sham-operated mice was blocked by AS-ODN for B2 receptors and that in injured mice by AS-ODN for B1 receptors. Altogether, these findings confirm a switching of receptor and fiber subtype for BK nociception after peripheral nerve injury, which might contribute to the pathobiology of neuropathic pain.

Animals↗

PubMed2003

Locus-specific rescue of GluRepsilon1 NMDA receptors in mutant mice identifies the brain regions important for morphine tolerance and dependence.

Tolerance and physical dependence caused by chronic treatment of narcotics are good models to study basic neuronal plasticity. Activation of the NMDA subtype of the glutamate receptor has been implicated as an anti-opioid system in the development of morphine analgesic tolerance and dependence. The present study examines the specific role of the epsilon1 subunit of the NMDA receptor using mice lacking the gene encoding epsilon1 subunit of the NMDA receptor (GluRepsilon1-/- mice). GluRepsilon1-/- mice showed significant enhancement and prolongation of morphine anti-nociception, compared with wild-type GluRepsilon1+/+ mice. GluRepsilon1-/- mice also showed a marked loss of the analgesic tolerance after repeated morphine treatments. In C57BL/6J mice treated with chronic morphine after tolerance paradigm, the GluRepsilon1 protein expression significantly increased in periaqueductal gray matter (PAG), ventral tegmental area (VTA) and nucleus accumbens (NAc), but not amygdala or hippocampus. The rescue of GluRepsilon1 protein by electroporation into the PAG and VTA, but not NAc of GluRepsilon1-/- mice significantly reversed morphine analgesic tolerance liability. Similar attempts were also performed in the naloxone-precipitated physical dependence paradigm. GluRepsilon1-/- mice showed marked loss of typical withdrawal abstinence behaviors, and significant enhancement of GluRepsilon1 protein expression was only observed in NAc by chronic morphine treatments after dependence paradigm. The rescue of GluRepsilon1 protein by electroporation into the NAc of GluRepsilon1-/- mice significantly reversed the loss of abstinence behaviors. These findings suggest that GluRepsilon1 has locus-specific roles in the development of morphine analgesic tolerance and physical dependence.

Animals↗

PubMed2003

The algogenic-induced nociceptive flexion test in mice: studies on sensitivity of the test and stress on animals.

Recently we developed a new technique, known as peripheral nociception test or algogenic-induced nociceptive flexion (ANF) test, to study the in vivo signal transduction of pain at the peripheral nerve endings in mice. In the present report, we examined the sensitivity of the method to detect pain signal and the stresses induced by the test on experimental animals. In the algogenic-induced biting and licking (ABL) test, bradykinin could not induce significant biting-licking response even at a dose of 1nmol. It induced significant biting-licking response only at 10nmol. However, with the ANF test, 100fmol of bradykinin was enough to produce sharp and significant nociceptive flexion response. Similarly, substance P, ATP and ONO-54918-07, a stable prostaglandin I(2) agonist, induced nociceptive flexion response in ANF test at much lower doses than needed to induce biting-licking responses in ABL test. Next, we measured the plasma corticosterone level after different nociception tests, which is a measure of stress on animals due to experimental manipulations. However, no significant rise in corticosterone level was observed with ANF test. Altogether, these findings indicate that the ANF test is a highly sensitive and less stressful technique to study in vivo mechanisms of pain at the peripheral nerve ending.

Adenosine Triphosphate↗

PubMed2003

Increased expression of vanilloid receptor 1 on myelinated primary afferent neurons contributes to the antihyperalgesic effect of capsaicin cream in diabetic neuropathic pain in mice.

Topical capsaicin is believed to alleviate pain by desensitizing the vanilloid receptor 1 (VR1) at the peripheral nerve endings. Here, we report that an up-regulation of VR1 expression on myelinated fibers contributes to the antihyperalgesic effect of capsaicin cream in streptozotocin (STZ)-induced diabetic neuropathic pain. Intravenous injection of STZ (200 mg/kg) in mice caused rapid onset of diabetes within 24 h. Thermal and mechanical hyperalgesia developed by 3 days after STZ injection and persisted at all time points tested until 28 days. There was also hyperalgesic response to intraplantar (i.pl.) prostaglandin I2 (PGI2) agonist-induced nociception in such mice. Application of capsaicin cream dose dependently reversed the thermal, mechanical, and PGI2 agonist-induced hyperalgesia observed in the diabetic mice. The i.pl. injection of capsaicin solution (0.4 microg/20 microl) produced nociceptive biting-licking responses in control mice, and these responses were significantly increased in STZ-induced diabetic mice. After neonatal capsaicin-treatment, which destroys most unmyelinated C-fibers, the i.pl. capsaicin-induced biting-licking responses were almost abolished. However, in neonatal capsaicin-treated diabetic mice, the i.pl. capsaicin-induced biting-licking responses reappeared. The i.pl. capsaicin-induced biting-licking responses were blocked by the competitive VR1 antagonist capsazepine. All these results suggest an increase in capsaicin receptor on myelinated fibers due to diabetes. Finally, we confirmed the up-regulation of VR1 expression on myelinated primary afferent neurons of diabetic mice by immunohistochemistry. Together, our results suggest that increased expression of VR1 on myelinated fibers might contribute to the antihyperalgesic effect of topical capsaicin in diabetic neuropathic pain.

Afferent Pathways↗

PubMed2003

Nocistatin and prepro-nociceptin/orphanin FQ 160-187 cause nociception through activation of Gi/o in capsaicin-sensitive and of Gs in capsaicin-insensitive nociceptors, respectively.

Nociceptin/orphanin FQ (N/OFQ), nocistatin, and prepro-N/OFQ 160-187 (C-peptide) are all derived from the same precursor protein. We examine the pharmacological mechanisms of nocistatin- and C-peptide-induced pronociceptive responses in a novel algogenic-induced nociceptive flexion test in mice. The intraplantar (i.pl.) injection of nocistatin- and C-peptide induced pronociceptive responses in a range of 0.01 to 10 or 1 pmol, respectively, which showed 100- to 1000-fold less potent effects than the N/OFQ. The nociceptive effects of both peptides were not affected by 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazole-2-one (J-113397) (i.pl.), an N/OFQ receptor antagonist, indicating that they are mediated by a novel mechanism independent of activation of N/OFQ receptor. Like N/OFQ, nocistatin-induced nociception was abolished by i.pl. injection of pertussis toxin, phospholipase C inhibitor, or CP-99994, a neurokinin 1 receptor antagonist, indicating that nocistatin may elicit nociception through a substance P release from nociceptor endings via activation of Gi/o and phospholipase C. The nociception was abolished by neonatal pretreatment (s.c.) with capsaicin or by i.t. pretreatment with CP-99994, but not MK-801 (i.t.), an N-methyl-d-aspartate receptor antagonist. In contrast, C-peptide-induced nociception was attenuated by the pretreatment with antisense oligodeoxynucleotide for Galphas (i.t.) and with KT-5720 (i.pl.), a cyclic AMP-dependent protein kinase inhibitor, but not with pertussis toxin. The nociception was neither attenuated by neonatal capsaicin nor by i.t. injection with CP-99994, but it was attenuated by i.t. injection with MK-801. These results suggest that nocistatin and C-peptide derived from prepro-N/OFQ stimulate distinct nociceptive fibers through different in vivo signaling mechanisms.

Analgesics, Opioid↗

PubMed2003

In vivo pain-inhibitory role of nociceptin/orphanin FQ in spinal cord.

Because nociceptin/orphanin FQ (N/OFQ) has both pronociceptive (hyperalgesia) and antinociceptive actions in pharmacological experiments, and there is no significant difference in the nociceptive responses between NOP(-/-) mice and their wild-type (NOP(+/+)) littermates, the physiological role of N/OFQ in pain regulation remains to be determined. Under the hypothesis that the use of molecularly distinct nociception test may reveal the pain modality-specific role of N/OFQ, we attempted to examine the physiological role of N/OFQ in pain transmission by using newly developed algogenic-induced nociceptive flexion test in NOP(-/-) and NOP(+/+) mice or NOP antagonist-treated mice. The nociceptive flexor responses upon intraplantar injection of bradykinin or substance P, which stimulates polymodal substance P-ergic fibers, were markedly potentiated in NOP(-/-) mice, compared with those in its NOP(+/+) mice. However, there were no significant changes in NOP(-/-) mice with adenosine triphosphate or prostaglandin I(2) agonist, which stimulates glutamatergic but not substance P-ergic fibers. The nocifensive responses induced by substance P (i.t.) were also potentiated in NOP(-/-) mice. On the other hand, there were no significant differences in NK1-like immunoreactivity, [(3)H]substance P binding, or NK1 gene expression in the dorsal horn of the spinal cord between NOP(-/-) and NOP(+/+) mice. In addition, NOP antagonists decreased the threshold in nociception tests driving spinal substance P neurotransmission. All these findings suggest that the N/OFQ-ergic neuron may play an in vivo inhibitory role on the second-order neurons for primary polymodal substance P-ergic fibers in the spinal cord.

Animals↗