Inhibitory effects of Sanguisorba officinalis root extract on HYBID (KIAA1199)-mediated hyaluronan degradation and skin wrinkling.

OBJECTIVES: Hyaluronan (HA), an important constituent of extracellular matrix in the skin, has many biological activities such as hydration that contributes to firmness and bounciness of the skin. We have reported that reduction in HA in the papillary dermis and over-expression of HYBID (HYaluronan Binding protein Involved in hyaluronan Depolymerization, alias KIAA1199 or CEMIP), a key molecule for HA degradation in skin fibroblasts, are implicated in facial skin wrinkling in Japanese and Caucasian women. However, little or no information is available for substances which inhibit the HYBID-mediated HA degradation. METHODS: Inhibition of Sanguisorba officinalis root extract and ziyuglycoside I, one of the components of Sanguisorba officinalis root extract, to the HYBID-mediated HA degradation was assessed by size-exclusion chromatography of HA depolymerized by stable transfectants of HYBID in HEK293 cells (HYBID/HEK293 cells) or normal human skin fibroblasts (Detroit 551 cells and NHDF-Ad cells). The HYBID mRNA and protein expression was examined by quantitative real-time PCR and immunoblotting in the skin fibroblasts treated with Sanguisorba officinalis root extract, and size distribution of newly produced HA was evaluated by preparing metabolically radiolabelled HA. A double-blind, randomized and placebo-controlled study was carried out in the 21 healthy Japanese women, who were topically treated with the formulation containing Sanguisorba officinalis root extract or the placebo on each side of the face including crow's foot area. RESULTS: Sanguisorba officinalis root extract, but not ziyuglycoside I, abolished HYBID-mediated HA degradation by HYBID/HEK293 cells. Sanguisorba officinalis root extract also inhibited HYBID-mediated HA degradation in skin fibroblasts by down-regulating HYBID mRNA and protein expression. Although control untreated skin fibroblasts produced polydispersed HA, the cells treated with Sanguisorba officinalis root extract produced only high-molecular-weight HA. Treatment with Sanguisorba officinalis root extract-formulated lotion significantly improved skin elasticity, and reduced skin wrinkling scores at the outer eye corner compared with the placebo formulation. CONCLUSION: Sanguisorba officinalis root extract showed an anti-HYBID-mediated HA degradation activity and anti-wrinkle activity on human facial skin, which is accompanied by the improvement in elasticity. Our study provides the possibility of a new strategy to inhibit HYBID-mediated HA degradation for anti-wrinkle care.

OBJECTIFS: l'acide hyaluronique (AH), un composant important de la matrice extracellulaire de la peau, assure de nombreuses activités biologiques, telles que l'hydratation qui contribue à la fermeté et l'élasticité de la peau. Nous avons rapporté que la réduction d'AH dans le derme papillaire et une surexpression de la protéine de liaison de l'AH impliquée dans la dépolymérisation de l'AH (HYBID, alias KIAA1199 ou CEMIP), une molécule clé de la dégradation de l'AH des fibroblastes cutanés, sont impliquées dans la formation des rides au niveau de la peau du visage chez les femmes d'origine japonaise et caucasienne. Cependant, peu ou aucune information n'est disponible concernant les substances qui inhibent la dégradation de l'AH provoquée par la protéine HYBID. MÉTHODES: l'inhibition de l'extrait de racine de la pimprenelle (Sanguisorba officinalis) et du ziyuglycoside I, l'un des composants de l'extrait de racine de Sanguisorba officinalis, sur la dégradation de l'AH provoquée par la protéine HYBID a été évaluée à l'aide d'une chromatographie par exclusion stérique de l'AH dépolymérisé par des transfectants stables de la protéine HYBID dans les cellules HEK293 (cellules HYBID/HEK293) ou les fibroblastes cutanés humains normaux (lignée cellulaire Detroit 551 et cellules des fibroblastes du derme humain chez l'adulte). L'expression de l'ARNm et de la protéine HYBID a été examinée par PCR quantitative en temps réel et par immuno-empreinte des fibroblastes cutanés traités avec de l'extrait de racine de Sanguisorba officinalis, et l'attribution des tailles des nouveaux échantillons produits de l'AH a été évaluée par préparation d'AH radiomarqué métaboliquement. Une étude en double aveugle, randomisée et contrôlée par placebo a été menée auprès des 21 femmes japonaises en bonne santé, qui ont été traitées localement avec la formulation élaborée à partir d'extraits de racine de Sanguisorba officinalis ou un placebo, sur chaque côté du visage, notamment sur la zone à pattes d'oie. RÉSULTATS: l'extrait de racine de Sanguisorba officinalis a permis d'arrêter la dégradation de l'AH provoquée par la protéine HYBID par les cellules HYBID/HEK293, mais ce n'était pas le cas du ziyuglycoside I. L'extrait de racine de Sanguisorba officinalis a également inhibé la dégradation de l'AH provoquée par la protéine HYBID des fibroblastes cutanés en diminuant l'expression de l'ARNm et des protéines HYBID. Bien que les fibroblastes cutanés témoins non traités aient produit de l'AH polydispersé, les cellules traitées aux extraits de racine de Sanguisorba officinalis ont produit uniquement de l'AH de haut poids moléculaire. Le traitement par lotion formulée à partir d'extraits de racine de Sanguisorba officinalis a amélioré de manière significative l'élasticité de la peau et réduit les scores de vieillissement du coin extérieur de la peau autour des yeux, par rapport à la formulation placebo. CONCLUSION: l'extrait de racine de Sanguisorba officinalis a démontré une action anti-dégradation de l'AH provoquée par la protéine HYBID et une activité antirides au niveau de la peau du visage humain, s'accompagnant d'une amélioration de l'élasticité. Notre étude fournit la possibilité d'une nouvelle stratégie pour inhiber la dégradation de l'AH provoquée par la protéine HYBID dans le cadre des soins antirides.

Anti-interleukin-6 receptor antibody prevents loss of bone structure and bone strength in collagen-induced arthritis mice.

OBJECTIVE: Rheumatoid arthritis (RA) is a disease that typically induces secondary osteoporosis, which increases the risk of bone fractures. Anti-interleukin-6 (IL-6) receptor antibody is used to treat RA; however, its effect on bone strength is not clear. Therefore, we investigated the influence of MR16-1, an anti-mouse IL-6 receptor antibody, on bone structure and femoral strength in a collagen-induced arthritis (CIA) mouse model. METHOD: DBA/1J mice were immunized by intradermal injection of bovine type II collagen. MR16-1 was administered intraperitoneally at the same time as immunization. Thirty-five days after the first immunization, bone structure and bone strength were measured by micro-computed tomography and the three-point bending test. RESULTS: In the CIA group, most bone mineral density and bone structure parameters in the foot, femur, and lumbar spine were significantly lower than in the normal group. Moreover, the maximum load of the femoral shaft in the CIA group was significantly lower than in the normal group. MR16-1 treatment significantly prevented the CIA-induced deterioration of bone structure and loss of bone strength. CONCLUSION: These results suggest that CIA systemically induces a deterioration of bone structure and loss of bone strength, and that IL-6 signalling plays an important role in these processes.

Clinical outcome after 36 months of treatment with injections of autologous blood for recurrent dislocation of the temporomandibular joint.

We investigated the prognosis after three years of treatment for recurrent dislocation of the temporomandibular joint with autologous blood given intravenously in 21 patients with a mean (range) age 64 (17-92) years of whom 16 had coexisting systemic disease. The mean (range) follow up from the first injection was 64 (41-99) months. Eighteen patients had no recurrence during the first 36 months after their first injection, which showed that this minimally-invasive treatment was effective, particularly for those who had conditions that made a mouthpiece or operation unsuitable.

Complementary Indicators for Diagnosis of Hepatic Vein Stenosis After Pediatric Living-donor Liver Transplantation.

INTRODUCTION: Although hepatic vein stenosis after liver transplantation is a rare complication, the complication rate of 1% to 6% is higher in pediatric living-donor liver transplantation than that in other liver transplantation cases. Diagnosis is very important because this complication can cause hepatic congestion that develops to liver cirrhosis, graft loss, and patient loss. However, this is unlikely in cases where there are no ascites or hypoalbuminemia. OBJECTIVES: Eleven of 167 patients who had undergone pediatric living-donor liver transplantation were identified in the outpatient clinic at Jichi Medical University as having suffered from hepatic vein stenosis, and were enrolled in the study. METHODS: We conducted a retrospective study in which we reviewed historical patient records to investigate the parameters for diagnosis and examine treatment methods and outcomes. RESULTS: The 11 patients were treated with 16 episodes of balloon dilatation. Three among these received retransplantation and another 2 cases required the placement of a metallic stent at the stenosis. Histological examination revealed severe fibrosis in four of nine patients who had a liver biopsy, with mild fibrosis revealed in the other five grafts. Furthermore, hepatomegaly and splenomegaly diagnosed by computed tomography, elevated levels of hyarulonic acid, and/or a decrease in calcineurin inhibitor clearance were found to be pathognomonic at diagnosis, and tended to improve after treatment. CONCLUSIONS: Diagnosis of hepatic vein stenosis after liver transplantation can be difficult, so careful observation is crucial to avoid the risk of acute liver dysfunction. Comprehensive assessment using volumetry of the liver and spleen and monitoring of hyarulonic acid levels and/or calcineurin inhibitor clearance, in addition to some form of imaging examination, is important for diagnosis and evaluation of the effectiveness of therapy.

Blockade of interleukin-6 receptor enhances the anti-arthritic effect of glucocorticoids without decreasing bone mineral density in mice with collagen-induced arthritis.

In a mouse arthritis model, we investigated whether interleukin-6 receptor (IL-6R) blockade would enhance the anti-arthritic effect of glucocorticoids (GCs). DBA/1J mice were immunized with type II collagen (CII), and were treated with prednisolone (PSL) and/or anti-mouse IL-6R antibody (MR16-1). Also, the effects of IL-6 on gene expression and the nuclear translocation of glucocorticoid receptors (GRs) were examined in cultured cells treated with dexamethasone (DEX). PSL reduced the arthritis score dose-dependently in the collagen-induced arthritis (CIA) mouse model. The arthritis score in the PSL (3 mg/kg) + MR16-1 group was lower than in the PSL (3 mg/kg) group, and at the same level as in the PSL (6 mg/kg) group. Lumbar vertebra bone mineral density (BMD) was decreased significantly in CIA mice and was higher in the PSL (3 mg/kg) + MR16-1 group than in the PSL (6 mg/kg) group. In the in-vitro synovial cells, IL-6 pretreatment attenuated the inhibitory effect of DEX on cyclooxygenase (COX)-2 expression and inhibited the nuclear translocation of GR induced by DEX. In contrast, in MC3T3-E1 osteoblastic cells, IL-6 pretreatment exacerbated the decrease in expression of osteocalcin and the increase in expression of receptor activator of nuclear factor kappa-B ligand (RANKL) by DEX. We demonstrated that IL-6 signalling blockade by an anti-IL-6R antibody can augment the anti-arthritic effect of GCs and inhibit the bone loss they cause.

Randomized clinical trial: rikkunshito in the treatment of functional dyspepsia--a multicenter, double-blind, randomized, placebo-controlled study.

BACKGROUND: Rikkunshito, a standardized Japanese herbal medicine, is thought to accelerate gastric emptying and relieve dyspepsia, although no large-scale, randomized, placebo-controlled trials of rikkunshito have been conducted. This study aimed to determine the efficacy and safety of rikkunshito for treating functional dyspepsia (FD). METHODS: FD patients received 2.5 g rikkunshito or placebo three times a day for 8 weeks in this multicenter, randomized, placebo-controlled, parallel-group trial. The primary end point was the proportion of responders at 8 weeks after starting test drug, determined by global patient assessment (GPA). The improvement in four major dyspepsia symptoms severity scale was also evaluated. In addition, plasma ghrelin levels were investigated before and after treatment. KEY RESULTS: Two hundred forty-seven patients were randomly assigned. In the eighth week, the rikkunshito group had more GPA responders (33.6%) than the placebo (23.8%), although this did not reach statistical significance (p = 0.09). Epigastric pain was significantly improved (p = 0.04) and postprandial fullness tended to improve (p = 0.06) in the rikkunshito group at week 8. Rikkunshito was relatively more effective among Helicobacter pylori-infected participants (rikkunshito: 40.0% vs placebo: 20.5%, p = 0.07), and seemed less effective among H. pylori-uninfected participants (rikkunshito: 29.3% vs placebo: 25.6%, p = 0.72). Among H. pylori-positive individuals, acyl ghrelin levels were improved just in rikkunshito group. There were no severe adverse events in both groups. CONCLUSIONS & INFERENCES: Administration of rikkunshito for 8 weeks reduced dyspepsia, particularly symptoms of epigastric pain and postprandial fullness. (UMIN Clinical Trials Registry, Number UMIN000003954).

Intra-arterial therapy with cisplatin suspension in lipiodol and 5-fluorouracil for hepatocellular carcinoma with portal vein tumour thrombosis.

BACKGROUND: Portal vein tumour thrombosis is a negative prognostic factor for hepatocellular carcinoma (HCC). AIM: To assess the efficacy of cisplatin in lipiodol emulsion combined with 5-fluorouracil (5-FU) for patients with HCC and portal vein tumour thrombosis. METHODS: The study subjects were 51 patients with the above-specified criteria who received injection of cisplatin suspension in lipiodol emulsion followed by intra-arterial infusion of 5-FU. The primary objective was to determine tumour response to the treatment, while the secondary objectives were safety and tolerability. Independent factors for survival were also assessed. RESULTS: Ten patients had complete response and 34 patients had partial response (response rate, 86.3%). The median survival for all 51 patients was 33 months, while that for 10 complete response patients and 21 patients who showed disappearance of HCC following additional therapies was 39 months. The single factor that significantly influenced survival was therapeutic effect. Treatment was well tolerated and severe toxicity was infrequent, with only grade 3 toxicity (thrombocytopenia) in one patient. CONCLUSIONS: The present study demonstrated the efficacy of hepatic arterial infusion chemotherapy using cisplatin-lipiodol emulsion and 5-FU without serious adverse effects in patients with unresectable HCC and portal vein tumour thrombosis.

Preseasonal prophylactic treatment with antihistamines suppresses nasal symptoms and expression of histamine H1 receptor mRNA in the nasal mucosa of patients with pollinosis.

Administration of antihistamines 2-4 weeks before the pollen season showed a greater inhibitory effect on nasal allergy symptoms in patients with seasonal allergic rhinitis. However, the mechanism of slow-onset effects of preseasonal treatment with antihistamines remains unclear. Here, we investigated the effect of preseasonal prophylactic treatment with antihistamines on nasal symptoms and the expression of histamine H1 receptor (H1R) mRNA of the nasal mucosa in patients with cedar pollen pollinosis. During the peak pollen period, the expression of H1R mRNA in the nasal mucosa and the scores of sneezing and watery rhinorrhea in patients receiving preseasonal prophylactic treatment with antihistamines were significantly suppressed in comparison with those in the patients without treatment. Moreover, there was a significant correlation between the nasal symptoms and the expression of H1R mRNA in both patients with or without preseasonal prophylactic treatment. These findings suggest that preseasonal prophylactic treatment with antihistamines is more effective than on-seasonal administration to patients with pollinosis in reducing nasal symptoms during the peak pollen period by suppressing H1R gene expression in the nasal mucosa.

Double-stranded RNA induces the synthesis of retinoic acid-inducible gene-I in vascular endothelial cells.

Viral infection induces various responses in vascular endothelial cells. Polyinosinic-polycytidylic acid (poly IC) is a synthetic double-stranded RNA (dsRNA), and treatment of cells with poly IC mimics the viral infection to the cells. Retinoic acid-inducible gene-I (RIG-I) is a protein belonging to the DExH-box family and designated as a putative RNA helicase. RIG-I is considered to play a role in antiviral responses through the regulation of gene expressions. In the present study, the authors treated human umbilical vein endothelial cells (HUVECs) with poly IC and found that poly IC induced the expression of RIG-I. The poly IC-induced RIG-I expression was inhibited by the preincubation of the cells with 2-aminopurine, an inhibitor of dsRNA-dependent protein kinase (PKR). Immunohistochemical examination revealed high levels of RIG-I immunoreactivity in vascular endothelial cells in the thalamus from rats inoculated with hantavirus. Induction of RIG-I by poly IC may be involved in the antiviral responses in endothelial cells.

Involvement of retinoic acid-inducible gene-I in the IFN-{gamma}/STAT1 signalling pathway in BEAS-2B cells.

Bronchial epithelial cells play an important role in airway host defence, and interferon (IFN)-gamma controls immune reactions by regulating the expression of various genes in bronchial epithelial cells. Signal transducer and activator of transcription 1 (STAT1) is the key transcriptional factor in IFN-gamma signalling. Retinoic acid-inducible gene-I (RIG-I) is a member of the DExH box family of proteins and designated a putative RNA helicase. RNA helicases play diverse roles in regulation of gene expression and cellular functions, and RIG-I is implicated in antiviral responses. The aim of the present study was to investigate the effect of IFN-gamma on RIG-I expression in a cell line derived from human bronchial epithelial cells, BEAS-2B. Induction of RIG-I in response to IFN-gamma was found in BEAS-2B cells. Induction of RIG-I by IFN-gamma was also demonstrated in another pulmonary epithelial cell line, NCI-H292. Transfection of BEAS-2B cells with RIG-I complementary DNA resulted in the upregulation of STAT1. Induction of IFN-gamma-inducible protein 10 by IFN-gamma was enhanced in the cells overexpressing RIG-I. It is concluded that retinoic acid-inducible gene-I may play an important role in the regulation of immunological reactions in bronchial epithelial cells elicited by interferon-gamma.

Hyperbaric oxygenation therapy for simple adhesive postoperative intestinal obstruction in children: comparison of the use of a short tube versus a long tube.

We hypothesized that hyperbaric oxygenation (HBO) combined with a long tube (LT) [HBO + LT] would be more effective than HBO combined with a short tube (ST) [HBO + ST] for simple adhesive postoperative intestinal obstruction (APIO) in children, assuming that there is synergism between HBO and LT. The objective of this study was to determine retrospectively the effect of HBO + LT compared to HBO + ST for simple APIO in children. Seventy-three patients were diagnosed with simple APIO, and 51 of these patients were treated with HBO + LT during 104 HBO sessions, while 22 were treated with HBO + ST during 34 HBO sessions. HBO was performed at a pressure of 2 atmospheres for 60 minutes once daily. The recovery rates after HBO therapy were 87.5 % for the HBO + LT group and 82.4 % for the HBO + ST group (p = 0.4496). HBO was performed 5 +/- 3 (range 1 to 15) times for the HBO + LT group and 4 +/- 2 (range 1 to 8) times for the HBO + ST group (p = 0.9847) for ultimate recovery from simple APIO. The recovery rate after HBO therapy of up to 6 sessions was 78.2 % for HBO + LT and 92.1 % for HBO + ST (p = 0.0360) among the cases that recovered ultimately. The effect of HBO + LT did not significantly differ from that of HBO + ST, but the insertion of an LT is more intricate and the cost is higher than that of an ST. Therefore, we conclude that the use of an LT is not necessarily required for HBO therapy for simple APIO.

Individual trait anxiety levels characterizing the properties of zen meditation.

Meditation is a specific consciousness state in which deep relaxation and increased internalized attention coexist. There have been various neurophysiological studies on meditation. However, the personal predispositions/traits that characterize the properties of meditation have not been adequately studied. We analyzed changes in neurophysiological parameters [EEG coherence and autonomic nervous activity using heart rate variability (HRV) as an index] during Zen meditation, and evaluated the results in association with trait anxiety (assessed by Spielberger's State-Trait Anxiety Inventory) in 22 healthy adults who had not previously practiced any form of meditation. During meditation, in terms of mean values in all subjects, an increase in slow alpha interhemispheric EEG coherence in the frontal region, an increase in high-frequency (HF) power (as a parasympathetic index of HRV), and a decrease in the ratio of low-frequency to HF power (as a sympathetic index of HRV) were observed. Further evaluation of these changes in individuals showed a negative correlation between the percent change (with the control condition as the baseline) in slow alpha interhemispheric coherence reflecting internalized attention and the percent change in HF reflecting relaxation. The trait anxiety score was negatively correlated with the percent change in slow alpha interhemispheric coherence in the frontal region and was positively correlated with the percent change in HF. These results suggest that lower trait anxiety more readily induces meditation with a predominance of internalized attention, while higher trait anxiety more readily induces meditation with a predominance of relaxation.

Cyclooxygenase-2 expression in thyroid neoplasms.

AIMS: Previous studies have demonstrated that cyclooxygenase-2 (COX-2) plays a role in carcinogenesis and carcinoma development. In this study, we investigated its expression in thyroid neoplasms in order to elucidate its role. METHODS AND RESULTS: COX-2 expression was studied immunohistochemically in 20 anaplastic (undifferentiated) carcinomas, 49 papillary carcinomas, 22 follicular carcinomas and 15 follicular adenomas. Positive staining was only occasionally seen in normal follicles or stromal cells. COX-2 over-expression was found in only 20.0% of follicular adenomas and 40.9% of follicular carcinomas. In papillary carcinomas, the incidence (81.3%) was significantly higher (P < 0.0001) than in follicular carcinomas, although COX-2 expression was reduced in cases with old age (P = 0.0190), large size (P = 0.0028), advanced stage (P = 0.0225), satellite tumours (P = 0.0363), and the presence of solid, scirrhous or trabecular growth patterns (P = 0.0018). Undifferentiated carcinomas less frequently over-expressed COX-2 (P = 0.0004), with an incidence of 40.0%. CONCLUSIONS: These results indicate that the up-regulation of COX-2 may contribute predominantly in the early phase of papillary carcinoma progression, whereas it plays a more adjuvant role in follicular carcinoma progression.

Enhancement of anti-cancer immunity by a lipoteichoic-acid-related molecule isolated from a penicillin-killed group A Streptococcus.

We isolated the lipoteichoic-acid-related molecule (OK-PSA) from OK-432, a streptococcal preparation, by affinity chromatography on CNBr-activated Sepharose-4B-bound monoclonal antibody TS-2, which neutralizes the interferon (IFN)-gamma-inducing activity of OK-432. We have previously reported that OK-PSA is a potent inducer of Th1-type cytokines in human peripheral blood mononuclear cells in vitro. In this study, we conducted an animal experiment to examine whether OK-PSA exhibits an anti-tumor effect in vivo by acting as a Th1 inducer in syngeneic Meth-A tumor-bearing BALB/c mice, in which the Th2 response is genetically dominant. It was found that OK-PSA induced Th1-type cytokines [IFN-gamma, tumor necrosis factor-alpha, interleukin (IL)-2, IL-12 and IL-18] in BALB/c mice bearing Meth-A tumor and caused a marked anti-tumor effect. Although it was suggested by an in vitro study. using spleen cells derived from the animals, that IL-18 plays the greatest role in the induction of the Th1-dominant state and tumor cell killing induced by OK-PSA, the in vivo experiments demonstrated that both IL-12 and IL-18 are essential in the anti-tumor effect exhibited by OK-PSA. These findings strongly suggest that OK-PSA is a major effector molecule of OK-432 and may be a useful immunotherapeutic agent, as a potent Th1 inducer, for cancer patients with a Th2-dominant state.

A case of renal ptosis treated with hochu-ekki-to with improvement confirmed by excretory urography.

A 59-year-old woman visited our clinic with complaints of right back dull pain. Excretory urography showed bilateral renal ptosis (a 6 cm decrease in position of the right kidney and a 5 cm decrease of the left kidney). She was treated with 7.5 g/day of Hochu-ekki-to. After 6 months, her symptoms improved and after 8 months excretory urography showed a 3 cm decrease in the position of both kidneys. Hochu-ekki-to might be useful for the conservative therapy of renal ptosis.

Three-dimensional structural model analysis of the binding site of lithocholic acid, an inhibitor of DNA polymerase beta and DNA topoisomerase II.

The molecular action of lithocholic acid (LCA), a selective inhibitor of mammalian DNA polymerase beta (pol beta), was investigated. We found that LCA could also strongly inhibit the activity of human DNA topoisomerase II (topo II). No other DNA metabolic enzymes tested were affected by LCA. Therefore, LCA should be classified as an inhibitor of both pol beta and topo II. Here, we report the molecular interaction of LCA with pol beta and topo II. By three-dimensional structural model analysis and by comparison with the spatial positioning of specific amino acids binding to LCA on pol beta (Lys60, Leu77, and Thr79), we obtained supplementary information that allowed us to build a structural model of topo II. Modeling analysis revealed that the LCA-interaction interface in both enzymes has a pocket comprised of three amino acids in common, which binds to the LCA molecule. In topo II, the three amino acid residues were Lys720, Leu760, and Thr791. These results suggested that the LCA binding domains of pol beta and topo II are three-dimensionally very similar.

Characteristics and distribution of endogenous RFamide-related peptide-1.

We have recently identified RFamide-related peptide (RFRP) gene that would encode three peptides (i.e., RFRP-1, -2, and -3) in human and bovine, and demonstrated that synthetic RFRP-1 and -3 act as specific agonists for a G protein-coupled receptor OT7T022. However, molecular characteristics and tissue distribution of endogenous RFRPs have not been determined yet. In this study, we prepared a monoclonal antibody for the C-terminal portion of rat RFRP-1. As this antibody could recognize a consensus sequence among the C-terminal portions of rat, human, and bovine RFRP-1, we purified endogenous RFRP-1 from bovine hypothalamus on the basis of immunoreactivity to the antibody. The purified bovine endogenous RFRP-1 was found to have 35-amino-acid length that corresponds to 37-amino-acid length in human and rat. We subsequently constructed a sandwich enzyme immunoassay using the monoclonal antibody and a polyclonal antibody for the N-terminal portion of rat RFRP-1, and analyzed the tissue distribution of endogenous RFRP-1 in rats. Significant levels of RFRP-1 were detected only in the central nervous system, and the highest concentration of RFRP-1 was detected in the hypothalamus. RFRP-1-positive nerve cells were detected in the rat hypothalamus by immunohistochemical analyses using the monoclonal antibody. In culture, RFRP-1 lowered cAMP production in Chinese hamster ovary cells expressing OT7T022 and it was abolished by pre-treatment with pertussis toxin, suggesting that OT7T022 couples G(i)/G(o) in the signal transduction pathway.

Xenon inhalation increases norepinephrine release from the anterior and posterior hypothalamus in rats.

PURPOSE: To investigate the effect of xenon (Xe) and nitrous oxide (N(2)O) on norepinephrinergic neuronal activity in the rat medial preoptic area (mPOA) and posterior hypothalamus (PH) using microdialysis. METHODS: Sixty male Wistar rats were equally allocated to two groups: mPOA and PH. A microdialysis probe was implanted into the mPOA or the PH. In both groups, each animal was exposed to one of the following inhalations: 25% oxygen (control, n=6), 30% Xe (n=6), 60% Xe (n=6), 30% N(2)O (n=6) or 60% N(2)O (n=6). Norepinephrine concentration in the perfused artificial cerebrospinal fluid was measured by high pressure liquid chromatography at ten-minute intervals. After plotting the time-norepinephrine concentration curve, the area under the curve (AUC) in each group was calculated. RESULTS: In the mPOA, 30 and 60% Xe, but only 60% N(2)O significantly increased norepinephrine release. The AUC in the 30% Xe, 60% Xe or 60% N(2)O group was 160 +/- 9 (P <0.05), 288 +/- 42 (P <0.01) or 237 +/- 46 pg x min/sample (P <0.01), respectively, compared to that in the control group: 77 +/- 14 pg x min/sample. In the PH, only 60% Xe significantly increased norepinephrine release compared to control (AUC: 191 +/- 38 vs. 71 +/- 1 pg x min/sample, P <0.01). CONCLUSION: The present data suggest that Xe stimulates norepinephrinergic neurons more potently than N(2)O; 1.2 times more in the mPOA and 2.5 times more in the PH. This stimulant effect may contribute to the hypnotic and sympathotonic effects of Xe in rats.

New ages for the last Australian megafauna: continent-wide extinction about 46,000 years ago.

All Australian land mammals, reptiles, and birds weighing more than 100 kilograms, and six of the seven genera with a body mass of 45 to 100 kilograms, perished in the late Quaternary. The timing and causes of these extinctions remain uncertain. We report burial ages for megafauna from 28 sites and infer extinction across the continent around 46,400 years ago (95% confidence interval, 51,200 to 39,800 years ago). Our results rule out extreme aridity at the Last Glacial Maximum as the cause of extinction, but not other climatic impacts; a "blitzkrieg" model of human-induced extinction; or an extended period of anthropogenic ecosystem disruption.

Evidence for a single nucleotide polymorphism in the KCNQ1 potassium channel that underlies susceptibility to life-threatening arrhythmias.

INTRODUCTION: Congenital long QT syndrome (LQTS) is a genetically heterogeneous arrhythmogenic disorder caused by mutations in at least five different genes encoding cardiac ion channels. It was suggested recently that common polymorphisms of LQTS-associated genes might modify arrhythmia susceptibility in potential gene carriers. METHODS AND RESULTS: We examined the known LQTS genes in 95 patients with definitive or suspected LQTS. Exon-specific polymerase chain reaction single-strand conformation polymorphism and direct sequence analyses identified six patients who carried only a single nucleotide polymorphism in KCNQ1 that is found in approximately 11% of the Japanese population. This 1727G>A substitution that changes the sense of its coding sequence from glycine to serine at position 643 (G643S) was mostly associated with a milder phenotype, often precipitated by hypokalemia and bradyarrhythmias. When heterologously examined by voltage-clamp experiments, the in vitro cellular phenotype caused by the single nucleotide polymorphism revealed that G643S-KCNQ1 forms functional homomultimeric channels, producing a significantly smaller current than that of the wild-type (WT) channels. Coexpression of WT-KCNQ1 and G643S-KCNQ1 with KCNE1 resulted in approximately 30% reduction in the slow delayed rectifier K+ current I(Ks) without much alteration in the kinetic properties except its deactivation process, suggesting that the G643S substitution had a weaker dominant-negative effect on the heteromultimeric channel complexes. CONCLUSION: We demonstrate that a common polymorphism in the KCNQ1 potassium channel could be a molecular basis for mild I(Ks) dysfunction that, in the presence of appropriate precipitating factors, might predispose potential gene carriers to life-threatening arrhythmias in a specific population.