Failure of parturition in mice lacking the prostaglandin F receptor.

Mice lacking the gene encoding the receptor for prostaglandin F2alpha (FP) developed normally but were unable to deliver normal fetuses at term. Although these FP-deficient mice showed no abnormality in the estrous cycle, ovulation, fertilization, or implantation, they did not respond to exogenous oxytocin because of the lack of induction of oxytocin receptor (a proposed triggering event in parturition), and they did not show the normal decline of serum progesterone concentrations that precedes parturition. Ovariectomy at day 19 of pregnancy restored induction of the oxytocin receptor and permitted successful delivery in the FP-deficient mice. These results indicate that parturition is initiated when prostaglandin F2alpha interacts with FP in ovarian luteal cells of the pregnant mice to induce luteolysis.

Hormone-induced apoptosis by Fas-nuclear receptor fusion proteins: novel biological tools for controlling apoptosis in vivo.

We have created fusion proteins between Fas and the ligand-binding domain of the estrogen or retinoic acid receptor. Murine fibrosarcoma L929 cells and human cervical carcinoma HeLa cells expressing the fusion proteins demonstrated apoptotic phenotypes in a tightly estrogen- or retinoic acid-dependent manner in vitro. Moreover, the fusion protein-expressing L929 cells transplanted into nude mice were also killed through apoptosis after injection of an estrogen agonist. This represents a novel system, "cell targeting," that can eliminate cells not only in vitro but also in vivo through the activation of a natural suicide machinery, i.e., apoptosis, by currently used hormones. This system implies wide applications not only in developmental biology and neurobiology but also in medicine, especially for cancer gene therapy.

Prostaglandin EP4 receptor agonist augments fixation of hydroxyapatite-coated implants in a rat model of osteoporosis.

The reduced stability of hydroxyapatite (HA)-coated implants in osteopenic conditions is considered to be a major problem. We therefore developed a model of a boosted cementless implantation in osteopenic rats.Twelve-week-old rats were either ovariectomised (OVX) or sham-operated (SO), and after 24 weeks plain or HA-coated implants were inserted. They were treated with either a prostaglandin EP4 receptor agonist (ONO-4819) or saline for one month. The EP4 agonist considerably improved the osteoporosis in the OVX group. Ultrastructural analysis and mechanical testing showed an improvement in the implant-bone attachment in the HA-coated implants, which was further enhanced by the EP4 agonist. Although the stability of the HA-coated implants in the saline-treated OVX rats was less than in the SO normal rats, the administration of the EP4 agonist significantly compensated for this shortage. Our results showed that the osteogenic effect of the EP4 agonist augmented the osteoconductivity of HA and significantly improved the stability of the implant-bone attachment in the osteoporotic rat model.

The regional distribution and cellular localization of mRNA encoding rat prostacyclin synthase.

The cloned cDNA for rat prostacyclin synthase was found to contain a 1503-bp open reading frame which encoded a 501-amino acid protein sharing 84.0% identity with the human enzyme. RNA blot analysis revealed that the rat prostacyclin synthase mRNA, as a single species of 2.1 kb, is expressed abundantly in the aorta and uterus. High levels of expression were also observed in the stomach, lung, heart, testis, liver, and skeletal muscle. Low but significant expression was also seen in the brain and kidney. Furthermore, the regional distribution and cellular localization of prostacyclin synthase mRNA were examined by in situ hybridization analysis of rat tissue sections. The definitive signals for the mRNA were localized in smooth muscle cells of the arteries, bronchi and uterus, and in the cells of the fibrous tunic surrounding the seminiferous tubules, which are characterized as smooth muscle cells. Besides smooth muscle cells, signals were also detected in the fibroblasts of the heart myocardium, lung parenchyma cells and kidney inner medulla tubules and interstitial cells.

Expression of messenger RNA for the prostaglandin D receptor in the leptomeninges of the mouse brain.

The localization of prostaglandin D receptor in the mouse brain was examined by in situ hybridization histochemistry. The autoradiography showed significant hybridization signals of mRNA for prostaglandin D receptor in the leptomeninges covering the surface of the brain, but not in neurons or glia in the brain parenchyma. This finding was confirmed by Northern blot analysis using mRNA prepared from either the whole brain with the leptomeninges, brain parenchyma without the leptomeninges or the leptomeninges alone. A weak signal corresponding to the major 3.5-kbp transcript was detected in the whole brain. This band was significantly enriched in the leptomeninges, but was not detected in the brain parenchyma. These results suggest that prostaglandin D receptor is most highly, if not exclusively, expressed in the leptomeninges of the mouse brain.

In situ hybridization studies of prostacyclin receptor mRNA expression in various mouse organs.

1. Expression of prostacyclin receptor (IP receptor) mRNA was examined in various mouse organs, and the cells expressing IP receptor mRNA were identified by in situ hybridization studies. Co-localization of mRNA for the IP receptor with that for preprotachykinin A (PPTA), a precursor protein for substance P, with mRNA for the prostaglandin E receptor subtypes (EP1, EP3 and EP4), and with renin mRNA, was examined by double in situ hybridization studies in the dorsal root ganglion and kidney, respectively. 2. IP receptor mRNA was expressed in the thymus and spleen. Expression in the thymus was found exclusively in the medulla, where mature thymocytes expressed transcripts for the IP receptor. Expression in the spleen was found as scattered signals over the white pulp and as punctate signals in the red pulp. The former was found in splenic lymphocytes and the latter in megakaryocytes. 3. IP receptor mRNA was also expressed in the vascular tissues of various organs such as the aorta, coronary arteries, pulmonary arteries and the cerebral arteries, where its expression was confined to smooth muscle cells. No expression was found in veins. In the kidney, IP receptor mRNA was detected in the interlobular arteries and glomerular arterioles but not in the juxtaglomerular (JG) cells which were labelled with the renin mRNA probe. 4. IP receptor mRNA was expressed in about 40% of the neurones in the dorsal root ganglion. Both small- and large-sized neurones were labelled but no labelling was found in the glia. Expression of PPTA mRNA was found in about 30% of total neurones. About 70% of these neurones expressed IP receptor mRNA, and about half of the IP receptor-positive neurones expressed PPTA mRNA. In addition to IP mRNA, mRNAs for EP1, EP3 and EP4 receptors were expressed in about 30%, 50% and 20%, respectively, of the dorsal root ganglion neurones. About 25%, 41% and 24% of the IP receptor-positive neurons co-expressed the EP1, EP3 and EP4 receptor, respectively. 5. These results not only verified IP receptor expression in various cells and tissues known to be sensitive to prostacyclin, but also revealed its expression in other systems, which urges the study of the actions of prostacyclin in these tissues. They also indicated that the actions of prostacyclin on blood vessels and platelets are mediated by the same type of receptor. Absence of IP receptor mRNA in the JG cells suggests that the action of prostacyclin on renin release may be indirect.

Transscleral fixation of acrylic intraocular lenses in the absence of capsular support through 3.5 mm self-sealing incisions.

PURPOSE: To evaluate the safety and efficacy of transscleral ciliary sulcus fixation of acrylic intraocular lenses (IOLs) through small incisions in the management of secondary IOL implantation. SETTING: Department of Ophthalmology, Osaka Rosai Hospital, Osaka, Japan. METHODS: This retrospective study consisted of 28 patients (30 eyes) who had transscleral fixation of acrylic IOLs through 3.5 mm incisions. All patients were followed for a minimum of 6 months in several different clinical settings. Data on visual acuity, keratometry, and central corneal endothelial cell count were evaluated preoperatively and postoperatively. The refractive error achieved and incidence of postoperative complications were determined. RESULTS: Uncorrected visual acuity (UCVA) improved in all eyes. Of the 18 eyes without pre-existing pathology, 11 (61.1%) had a UCVA of 20/40 or better from 1 week postoperatively. Best corrected visual acuity was unchanged in 24 eyes (80.0%) and improved by 2 Snellen lines or more in 5 eyes (16.7%) at the final examination. Self-sealing wound adaptation was achieved in 25 eyes (83.3%). The mean scalar shift in keratometric cylinder was 1.25 diopters (D) at 1 day postoperatively, 1.17 D at 1 week, and 1.06 D at 3 months. The rate of central corneal endothelial loss 6 months postoperatively averaged 7.84%. No intraoperative complications that were directly associated with acrylic IOL implantation occurred. Postoperative complications that included transient ocular hypertension, slight vitreous hemorrhage, and IOL malposition were found in a small population but resolved spontaneously without further surgical intervention. CONCLUSIONS: The good visual outcomes and low incidence of complications achieved in the present study indicate that acrylic IOLs positioned through small incisions might be considered for ciliary sulcus fixation. However, evaluation of this technique in a large population over the long term is required.

Intratumoral tumor necrosis factor induction and tumor growth suppression by ONO-4007, a low-toxicity lipid A analog.

BACKGROUND: ONO-4007 is a lipid A analog with low toxicity. MATERIALS AND METHODS: The antitumor activity and tumor necrosis factor (TNF)-inducing activity of ONO-4007 were compared with those of lipopolysaccharide (LPS) in WKAH rats bearing KDH-8 hepatoma cells. RESULTS: Weekly injections of ONO-4007 (3 and 10 mg/kg i.v.) suppressed tumor growth, but LPS (0.01 and 0.1 mg/kg i.v.) did not. A single injection of ONO-4007 (3 and 10 mg/kg i.v.) into tumor-bearing rats induced higher levels of endogenous TNF production in tumor tissues than LPS (0.001, 0.01 and 0.1 mg/kg i.v.). Repeated injections of LPS caused a reduction of TNF production in tumor tissues, whereas the reduction by ONO-4007 was less remarkable than that by LPS. Intratumoral injections of anti-rat TNF-alpha antibody attenuated the antitumor effect of ONO-4007. CONCLUSION: The antitumor effect of ONO-4007 is more pronounced than that of LPS and the effect is mediated by TNF produced in tumor tissues.

[Toxicity studies of landiolol hydrochloride (ONO-1101) (2). 4-week repeated dose intravenous toxicity study in rats with 4-week recovery test].

4-week repeated dose toxicity study with 4-week recovery test of landiolol hydrochloride (ONO-1101), a novel ultra short acting beta-blocker, was conducted in Sprague-Dawley (SD) rats. ONO-1101 was administered intravenously to rats of both sexes at a dose level of 0 (control), 12.5, 25, 50 or 100 mg/kg/day. In the 100 mg/kg/day group, bradypnea or dyspnea was seen in all animals, pale in ear, eye and foot, tremor, reddish lacrimation and loss of righting reflex were also observed in some animals right after administration, and then those signs disappeared within 1 min after administration. During the treatment period, 3/20 animals of each sex in the 100 mg/kg/day showed clonic convulsion and died within 2 min after administration. No clinical changes were seen in the 50 mg/kg/day group or lower. Histopathological findings showed atrophy of the submaxillary gland in females and vessel-wall thickening and perivascular fibrosis of the injection site (tail) in both sexes at 100 mg/kg/day, however those changes were reversible. ONO-1101 did not effect on body weight, food consumption, ophthalmology, urinalysis, hematology, blood chemistry, organ weights or necropsy at any doses. These results indicate that the no-adverse-effect level of ONO-1101 in rats is 50 mg/kg/day for both sexes in this study.

[Toxicity studies of landiolol hydrochloride (ONO-1101) (3). 4-week repeated dose intravenous toxicity study in dogs with 4-week recovery test].

4-week repeated dose toxicity study with 4-week recovery test of landiolol hydrochloride (ONO-1101), a novel ultra short acting beta-blocker, was conducted in beagle dogs. ONO-1101 was administered intravenously to dogs of both sexes at a dose level of 0 (control), 12.5, 25 or 50 mg/kg/day. No deaths occurred throughout the treatment period. Transitory licking chops, vomiting, nausea, diarrhea and soft feces were observed occasionally in both sexes dosed 25 and 50 mg/kg/day and the incidence seemed dose-dependent. However, those incidence declined in the course of the treatment period. Hematology showed a decrease in red blood cell count, hematocrit and hemoglobin value in both sexes receiving 25 and 50 mg/kg/day. ONO-1101 did not effect on body weight, food consumption, respiratory rate, pulse, rectal temperature, heart rate, blood pressure, electrocardiography, renal or hepatic function, ophthalmology, urinalysis, occult blood in feces, blood chemistry, organ weights, necropsy and microscopic findings at any doses. These results indicate that the no-adverse-effect level of ONO-1101 in dogs is 12.5 mg/kg/day for both sexes in this study.

[Toxicity study of sodium N-[2-[4-(2,2-dimethylpropionyloxy) phenylsulfonylamino] benzoyl] aminoacetate tetrahydrate (ONO-5046.Na) (3). 4-week repeated dose intravenous toxicity study in dogs with 4-week recovery test].

4-week repeated dose toxicity study with 4-week recovery test of sodium N-[2-[4-(2, 2-dimethylpropionyloxy) phenylsulfonylamino] benzoyl] aminoacetate tetrahydrate (ONO-5046.Na), a novel neutrophil elastase inhibitor, was conducted in beagle dogs. The dogs of both sexes were administered ONO-5046.Na intravenously at a daily dose of 0 (vehicle control), 7.5, 15 or 30 mg/kg. In the 15 mg/kg female group and the 30 mg/kg male and female groups, transient hypoactivity and ataxic gait were observed. It is considered that these symptoms were attributed to the pharmacological effect of ONO-5046.Na. Also, in the 30 mg/kg male and female groups, erythrocyte, hematocrit and hemoglobin were decreased. In the 30 mg/kg male group, lung weight was increased. However, histopathological examination revealed there were no changes in any organs including the lungs. There were no treatment-related changes in body weights, food consumption, ophthalmology, occult blood in feces, urinalysis, blood chemistry, electrocardiography, blood pressure, temperature, pulse rate, hepatic and renal function or necropsy. These results indicate that the NOAEL of ONO-5046.Na in dogs in 15 mg/kg/day for both sexes in this study.

[Toxicity study of sodium N-[2-[4-(2,2-dimethylpropionyloxy) phenylsulfonylamino] benzoyl] aminoacetate tetrahydrate (ONO-5046.Na) (2). 4-week repeated dose intravenous toxicity study in rats with 4-week recovery test].

A 4-week repeated dose toxicity study with 4-week recovery test of sodium N-[2-[4-(2,2-dimethylpropionyloxy) phenylsulfonylamino] benzoyl] aminoacetate tetrahydrate (ONO-5046.Na), a novel neutrophil elastase inhibitor, was conducted in Sprague-Dawley (SD) rats. The rats of both sexes were administered ONO-5046.Na intravenously at a daily dose of 0 (vehicle control and saline control), 18.75, 37.5, 75 or 150 mg/kg. ONO-5046.Na did not affect signs, body weight, food consumption, ophthalmology, urinalysis, hematology, blood chemistry, organ weights, necropsy or histopathology at any dose. These results indicate that the NOAEL of (ONO-5046.Na in rats is 150 mg/kg/day for both sexes in this study.

[Toxicity study of sodium N-[2-[4-(2,2-dimethylpropionyloxy) phenylsulfonylamino] benzoyl] aminoacetate tetrahydrate (ONO-5046.Na) (4). 6-month repeated dose intravenous toxicity study in rats with 1-month recovery test].

A 6-month repeated dose toxicity study with 1-month recovery test of sodium N-[2-[4-(2,2-dimethylpropionyloxy) phenylsulfonylamino] benzoyl] aminoacetate tetrahydrate (ONO-5046.Na), a novel neutrophil elastase inhibitor, was conducted in Sprague-Dawley (SD) rats. The rats of both sexes were administered ONO-5046.Na intravenously at a daily dose of 0 (vehicle control), 18.75, 37.5 or 75 mg/kg. ONO-5046.Na did not affect clinical signs, body weight, food consumption, opthalmology, urinalysis, hematology, blood chemistry, organ weight, necropsy or histopathology at any dose. These results indicate that the NOAEL of ONO-5046.Na in rats is 75 mg/kg/day for both sexes in this study.

Effect of allergen sensitization on external root resorption.

In orthodontic tooth movement (OTM), we should be concerned about external root resorption (ERR) as an undesirable iatrogenic problem, but its mechanisms are not fully understood. Since our previous epidemiologic studies found that patients with allergic diseases showed higher rates of ERR during orthodontic treatment, we explored the possible effect of allergic sensitization on ERR. In ovalbumin (OVA)-sensitized Brown-Norway rats, the amounts of ERR and OTM were greater than those in animals subjected to orthodontic force alone. The expression levels of RANKL and pro-inflammatory cytokines were increased in the periodontal tissues of sensitized rats with OTM, compared with control rats. Furthermore, leukotriene B4 (LTB4), a potent lipid mediator of allergic inflammation, and enzymes of the 5-lipoxygenase pathway, the biosynthetic pathway of leukotrienes, were also up-regulated. We found that low doses of aspirin suppressed ERR in allergen-sensitized rats, as well as the expressions of RANKL, pro-inflammatory cytokines, and LTB4. The present findings indicate that allergen sensitization has adverse effects on ERR under OTM, and that aspirin is a potential therapeutic agent for combating ERR.

cDNA cloning of a mouse prostacyclin receptor. Multiple signaling pathways and expression in thymic medulla.

A functional cDNA for a mouse prostacyclin receptor was isolated from a mouse cDNA library by reverse transcription polymerase chain reaction and hybridization screening. The cDNA encodes a polypeptide of 417 amino acid residues with putative seven transmembrane domains and an calculated molecular weight of 44,722. The amino acid sequence is 30-40% identical in the transmembrane domains to those of the mouse prostaglandin (PG) E receptor subtypes and thromboxane A2 receptor. [3H]Iloprost, a specific prostacyclin receptor radioligand, specifically bound to the membrane of Chinese hamster ovary cells permanently expressing the cDNA with Kd of 4.6 nM. This binding was displaced with unlabeled prostanoids in the order of cicaprost > iloprost, both prostacyclin agonists > PGE1 > carbacyclin >> PGD2 approximately STA2, a thromboxane A2 agonist approximately PGE2 > PGF2 alpha. Iloprost in a concentration-dependent fashion increased cAMP level and generated inositol phosphates in these cells, indicating that the receptor couples to multiple signal transduction pathways. Northern blot analysis revealed that the mRNA is expressed most abundantly in thymus, followed by spleen, heart, and lung. In situ hybridization of thymus showed that it is expressed exclusively in medulla and not in cortex.

Cellular localization of mRNAs for prostaglandin E receptor subtypes in mouse gastrointestinal tract.

Regional and cellular distribution of mRNAs for prostaglandin E (PGE) receptor subtypes was investigated in the mouse gastrointestinal tract by in situ hybridization. Strong signals for EP1 transcripts were detected in cells of the muscularis mucosae layer, especially in the body of the stomach. Intense signals for EP3 transcripts were detected in neurons of the myenteric ganglia throughout the tract. Moderate EP3 mRNA expression was also observed in fundic gland epithelial cells, except for surface mucous cells in the stomach. Expression of EP4 mRNA was moderate in surface epithelial cells of the corpus and in glands from the surface to the base of the antrum. Strong EP4 signals were observed in the epithelium in the duodenum, jejunum, and ileum. In the ileum, signals were only observed in the upper part of the villi. However, no or weak signals for EP2 transcripts were detected. These findings suggest that PGE2 modulates various gastric or intestinal functions via at least three different PGE receptors.

Distribution and diversity of symbiotic thermophiles, Symbiobacterium thermophilum and related bacteria, in natural environments.

Symbiobacterium thermophilum is a tryptophanase-positive thermophile which shows normal growth only in coculture with its supporting bacteria. Analysis of the 16S rRNA gene (rDNA) indicated that the bacterium belongs to a novel phylogenetic branch at the outermost position of the gram-positive bacterial group without clustering to any other known genus. Here we describe the distribution and diversity of S. thermophilum and related bacteria in the environment. Thermostable tryptophanase activity and amplification of the specific 16S rDNA fragment were effectively employed to detect the presence of Symbiobacterium. Enrichment with kanamycin raised detection sensitivity. Mixed cultures of thermophiles containing Symbiobacterium species were frequently obtained from compost, soil, animal feces, and contents in the intestinal tracts, as well as feeds. Phylogenetic analysis and denaturing gradient gel electrophoresis of the specific 16S rDNA amplicons revealed a diversity of this group of bacteria in the environment.

Patent ductus arteriosus and neonatal death in prostaglandin receptor EP4-deficient mice.

The physiological role of the prostaglandin E2 receptor EP4 subtype was investigated by generation of EP4-deficient-mice by gene targeting. Loss of the EP4 receptor was not lethal in utero, but most EP4 (-/-) neonates became pale and lethargic approximately 24 h after birth and died within 72 h. Less than 5% of the EP4 (-/-) mice survived and grew normally more than a year. Histological examination revealed that the ductus arteriosus in dead neonates remained open, while it was partially closed in the survivors. In situ hybridization study showed that EP4 mRNA was strongly expressed in the ductus. These results suggest that neonatal death is at least partly due to patent ductus arteriosus and that the EP4 receptor plays a role in regulation of the patency of this vessel. They also indicate that normal function of the EP4 receptor is essential in neonatal adaptation of the circulatory system.