Programmed necrosis, not apoptosis, is a key mediator of cell loss and DAMP-mediated inflammation in dsRNA-induced retinal degeneration.

There is no known treatment for the dry form of an age-related macular degeneration (AMD). Cell death and inflammation are important biological processes thought to have central role in AMD. Here we show that receptor-interacting protein (RIP) kinase mediates necrosis and enhances inflammation in a mouse model of retinal degeneration induced by dsRNA, a component of drusen in AMD. In contrast to photoreceptor-induced apoptosis, subretinal injection of the dsRNA analog poly(I : C) caused necrosis of the retinal pigment epithelium (RPE), as well as macrophage infiltration into the outer retinas. In Rip3(-/-) mice, both necrosis and inflammation were prevented, providing substantial protection against poly(I : C)-induced retinal degeneration. Moreover, after poly(I : C) injection, Rip3(-/-) mice displayed decreased levels of pro-inflammatory cytokines (such as TNF-α and IL-6) in the retina, and attenuated intravitreal release of high-mobility group box-1 (HMGB1), a major damage-associated molecular pattern (DAMP). In vitro, poly(I : C)-induced necrosis were inhibited in Rip3-deficient RPE cells, which in turn suppressed HMGB1 release and dampened TNF-α and IL-6 induction evoked by necrotic supernatants. On the other hand, Rip3 deficiency did not modulate directly TNF-α and IL-6 production after poly(I : C) stimulation in RPE cells or macrophages. Therefore, programmed necrosis is crucial in dsRNA-induced retinal degeneration and may promote inflammation by regulating the release of intracellular DAMPs, suggesting novel therapeutic targets for diseases such as AMD.

Growth and photosynthetic responses of two pine species (Pinus koraiensis and Pinus rigida) in a polluted industrial region in Korea.

We investigated the effects of pollutants on two pine species (Pinus koraiensis and Pinus rigida) in an industrial region in Korea, using a physiological approach. The concentrations of fluorine (F) and chlorine (Cl) in the atmosphere, in precipitation and soil water at the damaged site were all significantly higher than at a control site. Moreover, the concentrations of F, Cl and Mn in pine needles were significantly higher, and essential elements and chlorophyll in needles were significantly lower at the damaged site than at the control site. The photosynthetic capacities, shoot length and survival statistics of needles of the two pines were all significantly reduced at the damaged site compared to the control site, especially P. rigida. Based on our comparison of photosynthetic responses and the concentrations of F, Cl and Mn in needles of the two pine species, P. koraiensis is more resistant to excess Mn in its needles than P. rigida.

Effects of deicing salt on the vitality and health of two spruce species, Picea abies Karst., and Picea glehnii Masters planted along roadsides in northern Japan.

In northern Japan, the growth of Picea abies Karst., and Picea glehnii Masters, which have been planted along the highways, is often suppressed due to several environmental stresses. To examine the adverse effects of deicing salt, the primary source of stress,we measured needle life span, photosynthetic capacity, and water potential and transpiration rate of the two spruce species at a site with damaged trees, near the roadside and a site with healthy trees, located far from the highway. Results from the analysis showed large amounts of sodium and chlorine in the soil and snow at the damaged site. These elements had accumulated in the needles of the spruce. Moreover, physiological traits of the spruce, at the damaged site were also affected. Therefore, we concluded that poor physiological traits might be attributed to an accumulation of deicing salt in the needles, resulting in the suppression of tree growth.

Growth and annual ring structure of Larix sibirica grown at different carbon dioxide concentrations and nutrient supply rates.

We compared effects of ambient (360 vpm) and elevated (720 vpm) carbon dioxide concentration ([CO2]) and high and low nutrient supply rates on stem growth, annual ring structure and tracheid anatomy of Siberian larch (Larix sibirica Ledeb.) seedlings over two growing seasons. Elevated [CO2] had no significant effect on either stem height or diameter growth; however, both stem height and diameter growth were enhanced by the high nutrient supply rate, and these increases were stimulated by elevated [CO2]. Elevated [CO2] tended to increase the width of the annual xylem ring, the number of cells in a radial file spanning the ring, and tracheid lumen diameter, whereas it tended to reduce cell wall thickness, although there were no statistically significant CO2 effects on tracheid anatomy. Changes in tracheid cell morphology seemed to be dependent on changes in shoot elongation rates.

Japanese expert public health nurses empower clients with schizophrenia living in the community.

1. Home visiting psychiatric public health nurses are integral to expanding community-based mental health services for Japanese citizens living with schizophrenia and other mental illnesses. 2. Interviews with experienced psychiatric public health nurses revealed empowerment as the basic social process that guides their practice. Five empowering practice domains foster client autonomy. 3. Psychiatric public health nurses work with families, neighbors, educators, and employers to enable their clients' healthy living in the community.

[Effectiveness of home help service for psychiatric patients. The results of a model project in S city].

PURPOSE: The purpose of this study was to evaluate the effectiveness of a home help service project for psychiatric patients in S city. METHODS: To assess the effectiveness of the home help service, home help service providers at S city in 1997 were invited to hearings and the transcripts were examined qualitatively. Based on the results, interview questions were developed. We then interviewed 28 home help providers who were providing or had completed the service project, as well as care managers as of July 1998. Home help providers divided their patient stories retrospectively into three time frames: before starting home care, one month after starting providing care and at the present. Amount of care provided and patients' self care level were scored and averages ere compared for the three time frames. RESULTS: The analysis of the hearing data revealed parameters for the effectiveness of home help service for psychiatric patients to be as follows: 1) enhanced ability of daily living, such as taking meals, maintaining hygiene and shopping, (primary effectiveness: 5 items); and 2) developing social function, such as being motivated, maintaining personal relationship, extending social life, and decreasing family burden, (secondary effectiveness: 9 items). The amount of care needed was found to be significantly decreased and social function was significantly enhanced on comparisons between before starting home care and one month later, as well as between before starting home care and the present. DISCUSSION: Home help service for psychiatric patients is not only useful for enhancing patients' ability to succeed in daily living tasks, but also for developing their social functioning. Effectiveness was also identified in the level of patients' behavior. The results provide reasonable grounds for extended use of the home help service.

Radioimmunoassay for orexin A.

A radioimmunoassay for orexin A has been developed. Anti-orexin A antiserum was raised in New Zealand white rabbits immunized with a conjugate of synthetic orexin A with bovine serum albumin. This antibody did not crossreact with orexin B, hypothalamic hormones, pituitary hormones, neuropeptides or gut hormones. Radioiodination of orexin A was performed with the chloramin T method, followed by purification of radioiodinated material on Sephadex G-25 column. Orexin A was extracted from tissues using acid-acetone. The assay was performed with a double antibody system. The dilution curve of acid-acetone-extracts of rat hypothalamus in the radioimmunoassay system was parallel to the standard curve. The recovery of tissue orexin A was about 80%,and the intra-assay and inter-assay variations were 5.2% and 7.8%, respectively. Orexin A was found in the hypothalamus, cerebrum and testis. These data suggest that this assay system is suitable for the measurement of tissue orexin A and that orexin A is found in the central nervous system and testis.

Effects of orexin A on thyrotropin-releasing hormone and thyrotropin secretion in rats.

Effects of orexin A on secretion of thyrotropin-releasing hormone (TRH) and thyrotropin (TSH) in rats were studied. Orexin A (50 microg/kg) was injected iv, and the rats were serially decapitated. The effects of orexin A on TRH release from the rat hypothalamus in vitro and on TSH release from the anterior pituitary in vitro were also investigated. TRH and thyroid hormone were measured by individual radioimmunoassays. TSH was determined by the enzyme-immunoassay method. The hypothalamic TRH contents increased significantly after orexin A injection, whereas its plasma concentrations tended to decrease, but not significantly. The plasma TSH levels decreased significantly in a dose-related manner with a nadir at 15 min after injection. The plasma thyroid hormone levels showed no changes. TRH release from the rat hypothalamus in vitro was inhibited significantly in a dose-related manner with the addition of orexin A. TSH release from the anterior pituitary in vitro was not affected with the addition of orexin A. The findings suggest that orexin A acts on the hypothalamus to inhibit TRH release.

Gene amplification as a common cause of inherited thyroxine-binding globulin excess: analysis of one familial and two sporadic cases.

T4-binding globulin (TBG) is the major thyroid hormone transport protein in humans. Inherited abnormalities in the level of serum TBG have been classified as partial deficiency, complete deficiency and excess. A single nucleotide deletion or substitution in the TBG gene, located on Xq22, has been detected in partial and complete deficiencies. As for inherited TBG excess, the gene amplification has been recognized in two Japanese families recently. In this study, an additional three Japanese families, one familial (F-I) and two sporadic TBG excess (F-II, F-III), were analyzed. Serum TBG levels in hemizygous males were 73, 47 and 42 microg/ml, three- to two-fold the normal value. The molecule had normal properties in terms of heat stability and isoelectric focussing pattern. The gene dosage of TBG was evaluated by coamplification with autosomal betaGlobin or X-chromosomal Duchenne Muscular Dystrophy (DMD) and subsequent quantitation by HPLC. The TBG/betaGlobin ratios of the affected male and female of F-I were 3.09- and 3.86-times, respectively, compared to that of the normal males. The TBG/DMD ratios were 2.93- and 2.09-times, respectively. These results are compatible with three copies of the TBG gene on the affected X-chromosome. Similarly, a twofold increase in gene dosage was demonstrated in the affected males of sporadic cases. Their mothers with normal TBG values had the same TBG gene dosage as normal females, suggesting that de novo gene duplication arose in gametes probably during meiosis. Amplification of the TBG gene was not recognized in these three families by in situ hybridization of prometaphase chromosomes. Though the mechanism remains unproved, gene amplification of TBG was considered to be a common cause for inherited TBG excess.

Distribution of thyrotropin releasing hormone receptor type 2 in rats: an immunohistochemical study.

OBJECTIVE: To investigate the organ distribution of thyrotropin releasing hormone receptor (TRHR) type 2 in rats by immunohistochemical method. METHODS: TRHR type 2 was identified immunohistochemically in the rat tissues using specific anti-TRHR antiserum raised in New Zealand white rabbits immunized with a conjugate of synthetic TRHR type 2 (5-23) with bovine serum albumin. Immunohistochemical analysis was performed by avidin-biotin complex method. RESULTS: TRHR type 2 immunoreactivity was visualized in the central nervous system, anterior pituitary, gastric mucosa, Auerbach's and Meissner's nervous branch of the stomach, small intestine and colon, retina amd testis. Significant stain was detected in neural perikarya, axons and dendrites. When using antiserum preincubated with synthetic TRHR type 2(5-23) or anterior pituitary homogenates, no significant stain of anterior pituitary was detected. CONCLUSIONS: These findings suggest that TRHR type 2 is widely distributed and that the method used is valuable in studying the distribution of TRHR type 2 in rats.

Distribution of calcium sensing receptor in rats: an immunohistochemical study.

OBJECTIVE: To investigate the organ distribution of calcium sensing receptor (CaR) in rats by immunohistochemical method. METHODS: CaR was identified immunohistochemically in the rat tissues using specific anti-CaR antiserum raised in New Zealand white rabbits immunized with a conjugate of synthetic CaR peptide (186-204) with bovine serum albumin. Immunohistochemical analysis was performed by avidin-biotin complex method. RESULTS: CaR immunoreactivity was visualized in the central nervous system, anterior pituitary, gastric mucosa, small intestine and colon, Auerbach,s and Meissner,s gastric nervous branch, small intestine and colon, pancreas, adrenal medulla, kidney and testis. When using antiserum preincubated with synthetic CaR peptide (186-206) or kidney homogenates, no significant stain of kidney was detected. CONCLUSIONS: The findings suggest that CaR is widely distributed and that the method used is valuable in studying the distribution of caR in rat.

Nociceptin stimulates thyrotropin secretion in rats.

Effects of nociceptin on thyrotropin (TSH) and thyrotropin-releasing hormone (TRH) secretion in rats were studied. Nociceptin (150 microgram/kg) was injected intravenously and rats were serially decapitated after the injection. The effects of nociceptin on TRH release from the hypothalamus and TSH release from the anterior pituitary in vitro were also investigated. TRH and thyroid hormones were measured by individual radioimmunoassays. TSH was determined by enzyme immunoassay. TRH contents in the hypothalamus decreased significantly after nociceptin injection, whereas plasma TRH concentrations showed no changes. Plasma TSH concentrations increased significantly in a dose-related manner. The TRH release from the hypothalamus was enhanced significantly in a dose-related manner with the addition of nociceptin. The TSH release from the anterior pituitary in vitro was not affected by the addition of nociceptin. The plasma thyroxine and 3,3',5-triiodothyronine levels did not change significantly after nociceptin administration. The inactivation of TRH by plasma or hypothalamus in vitro after nociceptin injection did not differ from that of controls. The findings suggest that nociceptin acts on the hypothalamus to stimulate TRH and TSH secretion.

Thyrotropin-releasing hormone and somatostatin inhibit each others release in vitro in the rat retina.

The effect of thyrotropin-releasing hormone (TRH), somatostatin (SS) or octreotide, an analogue of SS, on release of TRH or SS from the rat retina was studied in vitro. The retina was incubated in medium 199 (pH 7.4) with 1.0 mg/ml of bacitracin (medium) for 20 min. The amount of TRH or SS release into the medium was measured by individual radioimmunoassays. The TRH release from the rat retina was inhibited significantly in a dose-related manner by the addition of SS or octreotide. The SS release from the retina was inhibited by TRH, and the inhibitory effect of TRH on SS release from the rat retina was blocked by the addition of anti-TRH receptor antiserum immunoglobulin fraction. The findings suggest an interaction between TRH and SS in the rat retina by which the addition of one inhibits the release of the other.

Biochemical and pharmacological characterization of YM-60828, a newly synthesized and orally active inhibitor of human factor Xa.

YM-60828 was found to potently inhibit human factor Xa following oral administration. YM-60828 showed high affinity for factor Xa (Ki = 1.3 nM), but did not affect thrombin (Ki > 100 microM). YM-60828 doubled factor Xa clotting time, prothrombin time (PT) and activated partial thromboplastin time (APTT) at 0.10, 0.21, 0.24 microM, respectively. Importantly, it did not prolong thrombin time at 100 microM. YM-60828 also inhibited factor Xa in the prothrombinase complex with an IC50 value of 7.7 nM. In addition to its anticoagulant activity, YM-60828 inhibited platelet aggregation induced by various agonists (IC50 = 3 to 23 microM). Squirrel monkeys were used to study the ex vivo anticoagulant activity and pharmacokinetic properties of YM-60828. One hour after oral administration at 3 mg/kg, YM-60828 strongly prolonged PT and APTT by 4.8- and 1.9-fold, respectively, and plasma concentration reached 788 +/- 167 ng/ml. Bioavailability was calculated to be 20.3%. These results strongly suggest that YM-60828 will be a valuable orally active and potent anticoagulant agent showing potential antithrombotic activity.

Radioimmunoassay for Aquaporin-2.

OBJECTIVE: To develop radioimmunoassay for aquaporin-2 (AQP-2). METHODS: Anti-AQP-2 antiserum has been raised in New Zealand white rabbits immunized with a conjugate of synthetic AQP-2 peptide (257-271) with bovine serum albumin. Radioiodination of synthetic peptide (tyrosine-AQP2 (257-271) was performed by chloramine T method, followed by purification of radioiodinated material on Sephadex G-25 column. RESULTS: The obtained antibody did not crossreact with vasopressin, pituitary hormones, hypothalamic hormones and neuropeptides. The assay was performed with a double antibody system. The values are expressed as an equivalent of synthetic AQP-2 peptide (257-271). The dilution curve of high AQP-2 urine in radioimmunoassay system was parallel to the standard curve. The recovery percentage of AQP-2 added to urine was about 100 % in this assay system. Intra-assay and inter-assay variation was 4.5 % and 7.2 %, respectively. Mean urinary excretion of AQP-2 was 1.16 ng equivalent of AQP-2 (257-271)/mg creatine and was lower in patients with diabetes insipidus. CONCLUSION: These data suggest that his assay system is a suitable to measure AQP-2 in urine.

The effects of Soybean Diet on Thyroid Hormone and Thyrotropin Levels in Aging Rats.

OBJECTIVE: To estimate the effect of soybean diet on serum level of thyroid hormone, its metabolites and thyrotropin (TSH) during aging in rats. METHODS: Male Donryu rats were fed laboratory chow containing 40 (Group A) or 10 volume percent (Group B) soybean protein, while controls (Group C) received regular laboratory chow. Groups of 10 animals of each groups were sacrificed by decapitation at the age of 12, 18, 24 and 30 months. Serum total thyroxine (T4), free thyroxine (FT4), 3,5,3'-triiodothyronine (T3), 3,3',5'-triiodothyronine (rT3) and 3,3'-diiodothyronine (3,3'-T2) and TSH concentrations were measured by specific radioimmunoassays. RESULTS: In Group A the level of T3 decreased significantly at from the age of 18 months, while in other groups such decrease was found only from the age of 24 months. Such changes were closely resembled by these in the level of 3,3'-T2, while inverse changes were observed in the level of rT3 which was increased in Group A from the age of 18 months and in the other groups from the age of 24 months. Serum T4 and FT4 level was decreased in all groups at the age of 30 months and no changes were observed in the level of TSH. CONCLUSIONS: The findings suggest that the level of T4, FT4 and T3 with its metabolite 3,3'-T2 stepwise decreased with aging, while that of rT3 showed inversely and increase. These changes were influenced by the content of soybean protein in the diet, the most rapid changes being found in the group with the high content of such protein.

Distribution of Dopamine Transporter in the Rat: an Immunohistochemical Study.

OBJECTIVE: To investigate the organ distribution of dopamine transporter (DAT) in rats by immunohistochemical method. METHODS: Dopamine transporter (DAT) was identified immunohistochemically in the tissues using specific antipeptide antiserum raised in New Zealand white rabbits immunized with a conjugate of synthetic DAT peptide (29-45) with bovine serum albumin. Immunohistochemical analysis was performed by avidin-biotin complex method. RESULTS: DAT immunoreactivity was visualized in the neural perikarya, axons and dendrites of the central nervous system, retina, adrenal medulla, Auerbach's nervous branch and Meissner's nervous branch of the stomach, small intestine and colon, anterior pituitary, and lung. When using antiserum preincubated with synthetic DAT peptide (DAT, 29-45) or hypothalamus homogenate which contains DAT, no significant stain of neurons in the hypothalamus was detected. CONCLUSION: These findings suggest that DAT is widely distributed and that the method used is valuable in studying the distribution of DAT in rats.

Purification and gas chromatographic-mass spectrometric characterization of non-methylene interrupted fatty acid incorporated in rat liver.

A C20 non-methylene interrupted trienoic acid detected in the liver of rat fed with a pine (Pinus koraiensis) seed oil diet was purified by two-step argentation thin-layer chromatography (AgTLC) and characterized by gas chromatography-mass spectrometry (GC-MS). First, a C20 methyl trienoate fraction was obtained from fatty acid methyl esters prepared from rat liver by 5% AgTLC developed with petroleum ether-diethyl ether-acetic acid (70:20:2, v/v) as a solvent system. The fraction was then subjected to AgTLC developed with benzene-acetone-diethyl ether-acetic acid (65:15:15:5, v/v) which could separate non-methylene interrupted fatty acids (NMIFA) from usual MIFAs. The purified C20 NMIFA was partially hydrogenated, and the resulting three kinds of the C20 monoenoate were analyzed by GC-MS after conversion to their dimethyl disulfide (DMDS) adducts. The results revealed that the original C20 non-methylene interrupted trienoic acid detected in the liver of rats fed with a pine seed oil diet was delta-5,11,14/20:3, a minor component of pine seed oil.

Effects of gamma-butyric acid on the release of thyrotropin-releasing hormone from the rat retina in vitro.

Effects of gamma-butyric acid (GABA) on the release of thyrotropin-releasing hormone (TRH) from the rat retina in vitro were studied. The rat retina was incubated in medium 199 (pH 7.4) with 1.0 mg/ml of bacitracin and 100 micrograms/ml of ascorbic acid (medium). The amount of TRH release into the medium was measured by radioimmunoassay. The TRH release from the rat retina was inhibited significantly in a dose-related manner with the addition of GABA, but not with bicuculline. The inhibitory effect of GABA on TRH release from the retina was blocked by adding bicuculline to the medium. The findings suggest that the GABAergic system inhibits TRH release from the rat retina in vitro.

Organ distribution of iodide transporter (symporter) in the rat: immunohistochemical study.

Iodide transporter/symporter (NIS) was identified immunohistochemically in rat tissues using specific antipeptide serum. Anti-NIS serum was raised in New Zealand white rabbits immunized with a conjugate of synthetic NIS peptide (39-53) with bovine serum albumin. Immunohistochemical analysis was performed by avidine-biotin complex method. NIS immunoreactivity was visualized in the thyroid gland, gastric and small intestine mucosa, anterior pituitary, adrenal medulla, pancreatic islets, kidney, chorioid plexus and several brain and spinal cord nuclei. When using antiserum preincubated with synthetic NIS peptide (39-53) or rat thyroid homogenate containing NIS, no significant stain of the thyroid gland was detected. These findings suggest that NIS is widely distributed and that the method used is suitable for studying the distribution of NIS in rats.