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.

Non-standard radionuclide production for PET in Japan.

There is a limited number of non-standard positron emission tomography (PET) radionuclides available in Japan. At the present time, non-standard PET nuclides ((64)Cu and (62)Zn/(62)Cu generator) are available from a medium energy cyclotron at the National Institute for Radiological Sciences in Chiba, Japan. Targetry for a small cyclotron has been installed on the cyclotrons of the University of Fukui. The production and distribution of these radionuclides from these cyclotrons will be described.

Selective ablation of porcine and rabbit liver tissue using radiofrequency: preclinical study.

Temperature changes and their distribution induced by 13.56-MHz radiofrequency (RF) heating of agar phantom and porcine and rabbit liver were investigated. It was possible to produce selective local heating of approximately 50 degrees C in the RF field of 2 x 2 x 2 cm(3) of the pig or rabbit liver. Coagulation necrosis after heating became pronounced and the margin between the coagulated lesion and normal tissue became clearer with time. Within 1 week after RF heating at 50 degrees C for 20 min, the coagulated area was replaced selectively and totally by necrotic tissue.

The accumulation and energy load of exogenous lipids in cirrhotic rat liver after partial hepatectomy.

BACKGROUND: The suitability of three energy substrates, glucose, medium-chain triglycerides (MCT) and long-chain triglycerides (LCT), was studied in cirrhotic rats after a partial hepatectomy. METHODS: Rats with thioacetamide-induced cirrhosis underwent a 70% hepatectomy, and were divided into three groups. Each group was then injected with 14C-labeled glucose, 14C-labeled MCT or 14C-labeled LCT, respectively. The subsequent tissue distribution of 14C and the cumulative amount of expired 14CO2 were determined. In a second experiment, the 70%-hepatectomized cirrhotic rats received total parenteral nutrition (TPN). The source of the nonprotein calories was 100% glucose (glucose group), 60% MCT + 40% glucose (MCT group), and 60% LCT + 40% glucose (LCT group). The adenylate energy charge and the glycogen content in the liver remnant were determined. RESULTS: The tissue distribution of 14C revealed that the fat emulsions accumulated preferentially in the liver. One hour after the partial hepatectomy, the concentration of 14C-labeled MCT in the liver remnants was threefold higher than in sham-operated controls. Similarly, the concentration of 14C-labeled LCT was twofold higher. The adenylate energy charge in the glucose group with TPN recovered to preoperative levels within 1-hour after the partial hepatectomy, whereas the LCT group with TPN showed a 24-hour delay in their recovery. The MCT group with TPN exhibited an intermediate time course. CONCLUSIONS: It is suggested that the specific accumulation of MCT and especially LCT emulsions in the cirrhotic liver remnant acts as an energy load rather than an energy substrate.

Absorption of a novel prodrug of L-dopa, L-3-(3-hydroxy-4-pivaloyloxyphenyl)alanine (NB-355). In vitro and in situ studies.

Absorption mechanism and absorption site of a prodrug of L-DOPA, L-3-(3-hydroxy-4-pivaloyloxyphenyl)alanine (NB-355, 1) was investigated using rats. Prodrug 1 (0.5 mM) was taken up by intestinal tissue segments time-dependently in vitro at pH 6.0. However, the rate of uptake was less than that of L-dopa. Inhibitors of the amino acid active transport system (L-Phe, dinitrophenol, ouabain) had no effect on the uptake of prodrug 1. In the intestinal tissue segments, prodrug 1 was extensively hydrolyzed by diisopropylfluorophosphate-sensitive esterase(s). To characterize the absorption site, gastrointestinal tracts were ligated to make acute loops in situ and prodrug 1 or L-dopa was injected into the loops. L-dopa disappeared rapidly from the lumen of the jejunum. In contrast, prodrug 1 disappeared rapidly from the ileum rather than the duodenum or jejunum. From these results, it was suggested that prodrug 1 was slowly absorbed primarily from the lower small intestine.

Quantification of L-3-(3-hydroxy-4-pivaloyloxyphenyl)alanine (NB-355) by high-performance liquid chromatography using o-phthalaldehyde/N-acetyl-L-cysteine derivatization.

A new and rapid high-performance liquid chromatographic assay has been developed for the determination of L-3-(3-hydroxy-4-pivaloyloxyphenyl)alanine (NB-355,I), a novel prodrug of L-DOPA. The method involves precolumn derivatization of the drug in biological samples with o-phthalaldehyde (OPA) and N-acetyl-L-cysteine (NAC) in a triethanolamine buffer (pH 8.0), giving a fluorescent compound that is stable for 2 h at 4 degrees C. Use of an internal standard improved the assay in accuracy and reliability. A programmable injector allowed automatic derivatization of large numbers of samples. Chromatographic separation was performed on a reversed-phase column (Capcell Pak C18) in which the silica gel was coated with silicone polymer. The peaks corresponding to compound I and the internal standard were eluted within 16 min with a mobile phase of acetonitrile-phosphate buffer (pH 7.1). The reliable limit of quantification was 0.5 pmol per injection (0.05 micrograms equivalents of L-DOPA per ml in plasma). The method was successfully applied for the measurements of dog plasma concentrations after oral dosing of compound I.

[Pharmacological studies of N-(2-mercapto-2-methylpropanoyl)-L-cysteine (SA96). V. Effects of SA96 in combination with indomethacin or prednisolone on adjuvant arthritis in rats].

SA96 in combination with indomethacin or prednisolone was investigated for their effects on the adjuvant arthritis in Lewis rats. SA96 given orally at a dose of 10 mg/kg inhibited the inflammation of adjuvant treated foot and untreated foot and the increase of serum Cu concentration which followed the development of adjuvant arthritis, but 2 mg/kg had no effect. Indomethacin given orally at a dose of 0.1 mg/kg or prednisolone given orally at a dose of 0.4 mg/kg also inhibited the adjuvant arthritis. Prednisolone suppressed the decrease of A/G ratio. Indomethacin and prednisolone, however, had no effects on the increase of serum Cu concentration. SA96 at a dose of 10 mg/kg in combination with indomethacin was more effective than the treatment of each drug alone on inflammation of adjuvant treated and untreated foot, serum Cu concentration, erythrocyte sedimentation rate and A/G ratio. The combination of SA96 at a dose of 2 mg/kg or 10 mg/kg with prednisolone had similar synergistic effects towards the adjuvant arthritis in rats.

[Pharmacological studies on N-(2-mercapto-2-methylpropanoyl)-L-cysteine(SA96). IV. Effects of SA96 and its main metabolite, SA679, on denaturation of human gamma-globulin and adjuvant arthritis in rats].

SA96 and its main metabolite, SA679, were investigated for their effects on denaturation of human gamma-globulin and bovine serum albumin (BSA) and on adjuvant arthritis in Lewis rats. The heat denaturation of human gamma-globulin and BSA enhanced by Cu2+ was inhibited by SA96, SA679 and D-penicillamine, and the inhibitory effect of SA96 was the most potent. In adjuvant arthritis rats, SA96 given orally at a dose of 10 mg/kg from the same day as the adjuvant injection inhibited significantly the swelling of adjuvant treated foot and untreated foot, increase of the relative organ weights of the adrenals, liver and kidney, and increase of serum Cu concentration; but at a dose of 100 mg/kg, it did not show any effects on these parameters. On the other hand, SA96 given from three days before the adjuvant injection showed the inhibitory effects at a dose of 100 mg/kg as well as 10 mg/kg. These results suggest that the effect of SA96 on adjuvant arthritis in rats depends on its administration schedule. SA679 also inhibited the adjuvant arthritis at a dose of 100 mg/kg, but its effect was less potent than that of SA96.

Effect of BR-227, a new bromhexine derivative, on secretory activities of tracheal secretory cells.

The effects of BR-227 on secretory activities of canine tracheal secretory cells including behavior of mucus glycoproteins were investigated histologically and histochemically. Following BR-227 treatment at a concentration of alcian blue at pH 2.5 and periodic acid-Schiff (AB (pH 2.5)/PAS) decreased in a concentration-dependent manner. Furthermore, a decrease in the thickness of the acini of submucosal glands and in marked increase in the ratio of acinar inner diameter of the gland to tracheal wall were induced after application of BR-227 at a concentration range of 10(-7) to 10(-4) M. The numbers of goblet and submucosal glandular cells which stained blue and purple with AB (pH 2.5)/PAS were decreased by BR-227 treatment in a concentrations-dependent way, whereas the cells which stained red were markedly increased. In the experiment using a combination of AB at pH 1.0 and PAS, a decrease in sulfated glycoproteins in those secretory cells was observed. Total saccharide and protein concentrations in the incubation fluid increased with BR-227 treatment, while N-acetylhexomsamine concentration tended to decrease. These findings suggest that BR-227 stimulates secretory activities of both goblet cells and submucosal glands, and lowers mucus viscosity in the secretory cells.

[Effect of brovanexine on secretory activities of tracheal secretory cells].

The expectorant effect of brovanexine (BvX) was investigated histologically and histochemically using isolated canine trachea. Following BvX treatment, the number of goblet cells (GC) stained positively with the combination procedure of alcian blue at pH 2.5 or pH 1.0 and periodic acid-Schiff was unaffected. The number of GC which stained blue and purple (stain index B & P) was reduced, while the number of GC that stained red (stain index R) was increased. The thickness of the acini of submucosal glands (SG) markedly decreased with 10(-5) and 10(-4)M BvX, and the ratio of the acinar inner diameter to the tracheal wall thickness significantly increased with 10(-4)M BvX. Stain index B & P of glandular cells decreased with 10(-5) and 10(-4)M BvX, which was accompanied by a marked increase in the stain index R. BvX treatment caused increases in total saccharide and protein concentrations in the incubation fluid, while N-acetylhexosamine slightly decreased. Both the BvX-induced histological changes in glandular cells and changes in concentrations of macromolecular components in the incubation fluid were much the same degree as those induced by bromhexine (Bh). The BvX-induced histochemical changes in secretory cells, however, were slighter than those induced by Bh. These findings suggest that BvX is an expectorant possessing both the secretagogic action selectively on SG and a mucolytic action toward acid glycoproteins in granules of secretory cells.

Pharmacological studies of 1-(2-chloro-4-hydroxyphenyl)-2-t-butylaminoethanol (HOKU-81), a new bronchodilator. 1st Communication: Bronchodilator and cardiovascular actions.

Bronchodilating action and influence on the cardiovascular system of 1-(2-chloro-4-hydroxyphenyl)-2-t-butylaminoethanol (HOKU-81), which is one of metabolites of tulobuterol obtained from rat urine, were examined using the isolated tracheae and atria of guinea pigs in vitro and dogs in vivo in comparison with those of the parent drug, i.e., tulobuterol isoprenaline, salbutamol and other reference bronchodilators. HOKU-81 was approximately 8 times more potent than tulobuterol, approximately twice as potent as salbutamol, and approximately as potent as isoprenaline in relaxing effect on the isolated tracheal smooth muscle preparation of guinea pigs. This effect of HOKU-81 seems to be due to direct action on the adrenergic beta-receptor. In anaesthetized dogs, bronchodilating effect of HOKU-81 was much more potent than that of tulobuterol and was approximately as potent as that of salbutamol when administered i.v., and its effect lasted for many hours. When administered orally, the duration of bronchodilating effect of HOKU-81 was almost as long as that of salbutamol. The cardiac stimulating effect of HOKU-81 examined in the isolated guinea pig artia and in anaesthetized dogs was weaker than those of isoprenaline and salbutamol, though stronger than that of tulobuterol. In the present studies in vitro as well as in vivo, the selectivity ratio of HOKU-81 for beta-adrenoceptors in the tracheal smooth muscle vs. those in the right atrial muscle was the largest of all the bronchodilators used.

Stimulation of methemoglobin reduction by selenium: a comparative study with erythrocytes of various animals.

The extent of stimulation of methemoglobin (metHb) reduction by selenite depends upon the level of reduced glutathione (GSH) in the erythrocytes. The reason for the species difference in the effect of selenite was discussed with respect to species differences in the GSH levels in erythrocytes.

Indirect hemagglutination test of Rickettsia orientalis: its specificity for reference strains, Karp, Gilliam and Kato.

Amberlite XE-64 and bovine serum albumin-treated rickettsia suspension, which was prepared from the chicken yolk sacs infected with each of 3 reference strains of Rickettsia orientalis, Karp, Gilliam and Kato, was sonicated at 10 KC for 15 min at 4 degrees C and centrifuged at 10,000 rpm for 40 min at 0 degrees C. The supernatant was used to sensitize the formalinized and tanned sheep red blood cells (SFTSRC). This antigen (2.5%) could be preserved for at least 1 wk at 4-8 degrees C and at least a month, if merthiolate (1:10,000) was added. Each SFTSRCP of 3 reference strains was found to be specific in indirect hemagglutination (IHA) reaction with each homologous immune serum and there was little cross reaction with the heterologous one. This minor cross reaction might be due to the presence of a small amount of soluble antigen in SFTSRC. No cross IHA reaction with the typhus fever immune serum was observed. The IHA test was considered to be useful for the diagnosis of scrub typhus.