Characterization of the Second Apoptosis Inhibitor Encoded by Guinea Pig Cytomegalovirus.

Despite the usefulness of guinea pig cytomegalovirus (GPCMV) for studies on congenital CMV infection, its viral mechanisms for the evasion of host defense strategies have not been fully elucidated. We reported previously that GPCMV gp38.1 functions as a viral mitochondria-localized inhibitor of apoptosis-like function, and its weak activity suggested the presence of an additional inhibitory molecule(s). Here, we identified gp38.3-2, a 42-amino-acid (aa) reading frame embedded within the gp38.3 gene that encodes a positional homolog of murine CMV (MCMV) m41. Characterization of gp38.3-2 resulted in the following findings: (i) the aa sequence of gp38.3-2 shows some similarity to that of MCMV m41.1, a viral inhibitor of oligomerization of a member of Bcl-2 family protein BAK, but there is no correspondence in their predicted secondary structures; (ii) gp38.3-2, but not gp38.3, showed inhibitory activities against staurosporine-induced apoptosis; (iii) three-dimensional protein complex prediction suggests that the N-terminal α-helix of gp38.3-2 interacts with residues in the BH3 and BH1 motifs of BAK, and analysis of gp38.3-2 and BAK mutants supported this model; (iv) guinea pig fibroblast cells infected with gp38.3-2-deficient GPCMV strain Δ38.3-2 died earlier than cells infected with rescued strain r38.3-2, resulting in lower yields of Δ38.3-2; (v) Δ38.3-2 exhibited a partial but significant decrease in monocyte and macrophage infection in comparison with r38.3-2; and, however, (vi) little difference in the viral infection of guinea pigs was observed between these two strains. Therefore, we hypothesize that gp38.3-2 contributes little to the evasion of host defense mechanisms under the experimental conditions used. IMPORTANCE Although GPCMV provides a useful animal model for studies on the pathogenesis of congenital CMV infection and the development of CMV vaccine strategies, our understanding of the viral mechanisms by which it evades apoptosis of infected cells has been limited in comparison with those of murine and human CMVs. Here, we report a second GPCMV apoptosis inhibitor (42 amino acids in length) that interacts with BAK, a Bcl-2 family proapoptotic protein. Three-dimensional structural prediction indicated a unique BAK recognition by gp38.3-2 via the BH3 and BH1 motif sequences. Our findings suggest the potential development of BH3 mimetics that can regulate inhibition or induction of apoptosis based on short ~40-amino-acid peptide molecules as with GPCMV.

Identification and functional analyses of a cell-death inhibitor encoded by guinea pig cytomegalovirus gp38.1 in cell culture and in animals.

Cytomegaloviruses (CMVs) employ an array of strategies designed to interfere with host defence responses against pathogens. Studies on such evasion mechanisms are important for understanding the pathogenesis of CMV diseases. Although guinea pig CMV (GPCMV) provides a useful animal model for congenital CMV infection, its evasion strategies are not fully elucidated. Here, we analysed a genome locus that may encode gene products for the GPCMV evasion mechanisms and found the following. (1) RACE analyses identified five transcripts in the GP38-gp38.4 locus, one of which was a spliced product encoding gp38.1. Similarities in the splicing pattern and gene position of gp38.1 to human CMV UL37 and its exon 1 encoding vMIA (viral mitochondria-localized inhibitor of apoptosis) suggest that the gp38.1 gene encodes an apoptosis inhibitor. (2) In a transient transfection assay, gp38.1 localized in the mitochondria and relocated BAX from the cytoplasm to the mitochondria, although its co-localization with BAK was not evident. Further, the expression of gp38.1 partially reduced staurosporine-induced apoptosis. (3) GPCMV defective in the gp38.1 ORF (Δ38.1) and the virus that rescues the defect (r38.1) were generated. Guinea pig fibroblast cells infected with Δ38.1 died earlier than r38.1-infected cells, which resulted in the lower yields of Δ38.1. (4) In animals, viral loads in the spleens of r38.1-infected guinea pigs were higher than those in the spleens of Δ38.1-infected animals. In conclusion, although GPCMV gp38.1 exerts a vMIA-like function, its inhibitory effect was not robust, suggesting the presence of additional inhibitory molecule(s), such as a BAK-specific inhibitor.

Biochemical evidence of cell starvation in diabetic hemodialysis patients.

Recently, the ratio of patients with diabetes mellitus (DM) among hemodialysis (HD) patients has increased to become the largest sub-population. Their prognoses are significantly worse than those of patients without diabetes (non-DM). In the present study, 10 DM patients who did not take meals and 10 non-DM patients who took meals during HD sessions were investigated. The time courses of the change in plasma levels of metabolites during HD were determined. DM patients exhibited decreased plasma levels of lactate, pyruvate and alanine and dramatically increased levels of ketone bodies. At the end of HD, the plasma levels of lactate, pyruvate, alanine and ketone body were 0.46 ± 0.07, 0.026 ± 0.01, 0.12 ± 0.04 and 0.26 ± 0.04 mM (mean ± standard error), respectively. The profile was 'hypolactatemia and hyperketonemia', indicating non-homeostasis. Glycolysis and tricarboxylic acid cycle were suppressed, and the oxidation of fatty acid was accelerated, indicating starvation, even though high amounts of glucose (150 mg/dl) in dialysate were supplied continuously to the bloodstream. In contrast, the plasma levels of lactate, pyruvate, and alanine in the non-DM patients were increased, with the levels of ketone body remaining low during HD to maintain homeostasis, indicating accelerated glycolysis. Furthermore, their plasma levels of insulin increased from 8.1 ± 1.4 to 19.8 ± 3.4 µU/ml, which indicated endogenous secretion stimulated by glucose in dialysate and meal intake. In contrast, in the DM patients, the levels decreased from 19.2 ± 3.4 to 5.5 ± 1.1 µU/ml. This value was the lower limit of the normal range. The depletion of the insulin through extracorporeal circulation may inhibit the transportation of glucose from the blood into the muscles, with the consequence of cell starvation. Such cell starvation along with lipolysis every two days may accelerate proteolysis and affect the prognosis of DM patients.

Plasma Levels of Biotin Metabolites Are Elevated in Hemodialysis Patients with Cramps.

Patients with renal failure undergoing hemodialysis (HD) are susceptible to muscle cramps during and after HD. Muscle cramps are defined as the sudden onset of a prolonged involuntary muscle contraction accompanied by severe pain. Through HD, water-soluble vitamins are drawn out with water. Since biotin, a water-soluble vitamin, plays an essential role as one of the coenzymes in producing energy, we have hypothesized that deficiency of biotin may be responsible for HD-associated cramps. We previously reported that biotin administration ameliorated the muscle cramps, despite the elevated plasma biotin levels before HD and biotin administration, as judged by an enzyme-linked immunosorbent assay (ELISA). However, the ELISA measures not only biotin but also total avidin-binding substances (TABS) including biotin metabolites. In the present study, we determined biotin in HD patients as well as healthy controls, using a newly developed method with ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The plasma samples were collected from 28 HD patients (16 patients with cramps and 12 patients without cramps) before HD and biotin administration and from 11 controls. The results showed that the accumulation of biotin and TABS in plasma of HD patients compared to controls. Importantly, the levels of biotin metabolites, i.e. TABS subtracted by biotin, increased significantly in patients with cramps over those without cramps. Moreover, the levels of biotin metabolites were significantly higher in patients with a poor response to administered biotin, compared to those with a good response. We propose that accumulated biotin metabolites impair biotin's functions as a coenzyme.

Efficacy of scallop shell powders and slaked lime for inactivating avian influenza virus under harsh conditions.

The efficacy and stability of scallop shell powder (SSP) were investigated, in terms of its capacity to inactivate avian influenza virus (AIV), and compared with slaked lime (SL). An environmental simulation was conducted by emulating sunlight and wet-dry conditions. The powders were collected at consecutive 2-week intervals under sunlight and upon every resuspension. These materials were tested by mixing them with AIV and incubating the mixture for 3 min or 20 h, followed by AIV titration. At the same time, a pH buffering test was conducted by neutralization with Tris-HCl. The results revealed that SSP and SL have high alkalinity and excellent ability to inactivate AIV. In a simulated harsh environment, SSP and SL retained a satisfactory ability to inactivate AIV within 20 h throughout the experimental procedure. However, SSP was able to inactivate AIV during a short contact period (3 min), even under harsh conditions, and it was more resistant than SL to neutralization.

Evaluation of sprayed hypochlorous acid solutions for their virucidal activity against avian influenza virus through in vitro experiments.

Hypochlorous acid (HOCl) solutions were evaluated for their virucidal ability against a low pathogenic avian influenza virus (AIV), H7N1. HOCl solutions containing 50, 100 and 200 ppm chlorine (pH 6) or their sprayed solutions (harvested in dishes placed at 1 or 30 cm distance between the spray nozzle and dish) were mixed with the virus with or without organic materials (5% fetal bovine serum: FBS). Under plain diluent conditions (without FBS), harvested solutions of HOCl after spraying could decrease the AIV titer by more than 1,000 times, to an undetectable level (< 2.5 log10TCID50/ml) within 5 sec, with the exception of the 50 ppm solution harvested after spraying at the distance of 30 cm. Under the dirty conditions (in the presence of 5% FBS), they lost their virucidal activity. When HOCl solutions were sprayed directly on the virus on rayon sheets for 10 sec, the solutions of 100 and 200 ppm could inactivate AIV immediately after spraying, while 50 ppm solution required at least 3 min of contact time. In the indirect spray form, after 10 sec of spraying, the lids of the dishes were opened to expose the virus on rayon sheets to HOCl. In this form, the 200 ppm solution inactivated AIV within 10 min of contact, while 50 and 100 ppm could not inactivate it. These data suggest that HOCl can be used in spray form to inactivate AIV at the farm level.

Tumor budding in colorectal carcinoma assessed by cytokeratin immunostaining and budding areas: possible involvement of c-Met.

Tumor budding/sprouting has been shown to be an independent adverse prognostic factor in T1 and T3N0 colorectal carcinomas, however, its assessment could be improved by more accurate identification of budding carcinoma cells and consideration of budding areas. Moreover, tumor budding mechanisms are yet to be defined. In this study, we evaluated the identification of budding tumor cells by either H&E staining alone or H&E with immunohistochemistry and developed a scoring system based on budding grades and areas. We examined whether the budding score correlated with clinicopathologic features and prognosis and the association between tumor budding/sprouting and c-Met protein expression and phosphorylation and MET gene copy numbers because c-Met is known to play an important role in colorectal carcinoma tumorigenesis. Cytokeratin immunohistochemistry could identify tumors with shorter disease-free survival (DFS) from the low-grade budding group assessed with H&E alone. High budding scores based on budding grade and area were more significantly correlated with DFS than scores obtained using the budding grade alone. In tumors with a high budding score, c-Met expression and phosphorylation levels and MET gene copy numbers were significantly increased at the invasive front compared with those in superficial tumor portions. This study showed for the first time that high levels of phospho-c-Met at the invasive front were significantly associated with a high budding score and shorter DFS. In conclusion, a budding score assessed by budding grades and budding-positive areas correlates highly with clinicopathologic aggressive features of colorectal carcinoma.

Inactivation of avian influenza virus, newcastle disease virus and goose parvovirus using solution of nano-sized scallop shell powder.

Scallop shell powder produced by calcination process - the average diameter of the powder particles being 20 µm (SSP) - was further ground into nano-sized particles, with average diameter of 500 nm, here designated CaO-Nano. Solution of CaO-Nano could inactivate avian influenza virus within 5 sec, whereas the solution of SSP could not even after 1 hr incubation. CaO-Nano solution could also inactivate Newcastle disease virus and goose parvovirus within 5 sec and 30 sec, respectively. The virus-inactivating capacity (neutralizing index: NI>3) of the solution was not reduced by the presence of 20% fetal bovine serum. CaO-Nano solution seems to be a good candidate of materials for enhancement of biosecurity in farms.

Inactivation of goose parvovirus, avian influenza virus and phage by photocatalyst on polyethylen terephthalate film under light emitting diode (LED).

The inactivation effect of a novel photocatalyst on polyethylene terephthalate film on goose parvovirus (GPV), avian influenza virus (AIV) and Qß phage was evaluated. Under a light emitting diode (LED) light (range 410-750 nm), GPV was inactivated by irradiation at 1,000 lux for 6 hr, while AIV and Qß phage were inactivated by irradiation at 150 lux for 2 hr. These data suggest that this new photocatalyst can potentially be used as one of the materials to inactivate viruses in the indoor environment and help us to prevent viral infectious diseases through indirect contact.

Characterization of silodosin and naftopidil in the treatment of bladder dysfunction in the spontaneously hypertensive rat.

PURPOSE: As increasing evidence suggest that α(1)-blockers prevent benign prostatic hyperplasia related overactive bladder and nocturia in the human, we investigated the effects of silodosin and naftopidil on hypertension-related bladder dysfunction in the spontaneously hypertensive rat (SHR) model. MATERIALS AND METHODS: Twelve-week-old male SHRs received no treatment or treatment with silodosin (100 µg/kg, p.o.) or naftopidil (10 or 30 mg/kg, p.o.) once daily for 6 weeks. Wistar rats were used as normotensive controls. After 6-week treatment, voiding functions were estimated by metabolic cages (dark- and light-cycle separately) and cystometric studies. Furthermore, the bladder blood flow (BBF) was measured employing the hydrogen clearance method. RESULTS: SHRs showed significant increases in micturition frequency, and decreases in BBF and single voided volume in both metabolic cages and cystometrograms compared to the Wistar group. Treatment with silodosin normalized the decreased BBF, and treatment with naftopidil increased the BBF in a dose-dependent manner in the SHR group. Although treatment with silodosin and the high dose of naftopidil significantly inhibited micturition frequency in one day, only treatment with the high dose of naftopidil significantly inhibited micturition frequency and urine production in the light-cycle compared to the non-treated SHRs. Although treatment with silodosin and the high dose of naftopidil significantly increased single voided volume, only treatment with silodosin significantly inhibited non-voiding contractions in the cystometrgrams. CONCLUSION: Our data suggest that both silodosin and naftopidil improve hypertension-related bladder dysfunction in the SHR, and naftopidil but not silodosin improves urinary frequency in the light-cycle due to inhibition of urine production.

Fasudil improves the endothelial dysfunction in the aorta of spontaneously hypertensive rats.

We investigated the effects of fasudil, a Rho kinase inhibitor, in the endothelial dysfunction of aortas from spontaneously hypertensive rats (SHRs). SHRs were divided in three groups; intraperitoneally (i.p.) vehicle-treated SHRs (SHR), SHRs treated with fasudil 3 mg/kg i.p. (Fas3), and SHRs treated with fasudil 10 mg/kg i.p. (Fas10). Vehicle-treated Wistar rats were used as normo-tensive control group. After a six-week-treatment, blood pressure and heart rate were measured by the tail cuff method. Afterwards animals were sacrificed and aortas were examined in vitro by organ bath studies to evaluate the contraction and relaxation ability. Rho kinase activity, myosin light chain (MLC), phosphorylated MLC (phospho-MLC), eNOS, phospho-eNOS protein expression and eNOS mRNA levels were evaluated. SHR demonstrated a significant hypercontractility and impaired relaxation compared to the control. Fasudil 10mg/kg significantly corrected the hypercontractility, restored the relaxation, and significantly decreased the mean arterial blood pressure, while no change observed in the systolic blood pressure. Rho kinase activity was significantly higher in the SHR, and was significantly inhibited by the high dose of fasudil. There was a slight up-regulation in the MLC, and phospho-MLC protein levels in the SHR. eNOS and phospho-eNOS protein levels were significantly lower in the SHR, and this abnormality was significantly normalized by fasudil treatment. No significant difference was observed in the eNOS gene expression. This study suggests that fasudil by inhibiting the Rho kinase activity normalizes the eNOS expression and phosphorylation and ameliorates the endothelial dysfunction induced by hypertension in the SHR model.

Antioxidant treatment with edaravone or taurine ameliorates diabetes-induced testicular dysfunction in the rat.

Diabetes mellitus with the subsequent generation of reactive oxygen species represents a major risk factor for testicular dysfunction (TD). We investigate whether administration of antioxidants edaravone and taurine could prevent type 1 diabetes-induced TD in the rat. Six-week-old male Wistar rats were divided into four groups. Group A was treated with citrate-phosphate buffer plus normal saline, whereas in the other three groups, diabetes was induced by streptozotocin (50 mg/kg intraperitoneally). Subsequently, the diabetic rats were treated for 4 weeks either with normal saline (group B), edaravone (10 mg/Kg/day, intraperitoneally; group C), or taurine (500 mg/Kg/day, intraperitoneally; group D). Body, testicular, and epididymal weight, serum glucose, malondialdehyde levels, 8-Hydroxy-2'-deoxyguanosine(8-OH-dG) levels, testicular catalase activity, and serum testosterone levels were determined. Histological examination and the Johnsen score were used to observe and evaluate, respectively, the morphological changes in the testes. TUNEL assay was used to examine DNA fragmentation. Mating studies were performed in order to evaluate the fertility potential of male rats in each group. Edaravone or taurine treatment prevented significantly the decreased body, testicular, and epididymal weight induced by diabetes. Moreover, edaravone or taurine significantly decreased the diabetes-induced malondialdehyde levels, 8-OHdG levels, the morphological damage, and the number of apoptotic cells. Taurine, but not edaravone, increased significantly the testicular catalase activity. The antioxidant treatment had no effect on the fertility potential of the diabetic rats. The morphological damage, increased lipid peroxidation, and apoptosis in testicular tissue can be significantly relieved by edaravone or taurine treatment through suppressing the increased oxidative stress in the rat testis.

Hydroxyfasudil ameliorates penile dysfunction in the male spontaneously hypertensive rat.

Hypertension represents a major risk factor for erectile dysfunction. Although the etiology of hypertension-induced erectile dysfunction is multifactorial and still unknown, Rho-Rho kinase pathway is one of the key factors. To investigate whether administration of hydroxyfasudil, a Rho kinase inhibitor could prevent dysfunction of NO-induced relaxation in corpus cavernosum smooth muscle in the SHR (spontaneously hypertensive rat), twelve-week-old male SHRs were treated with hydroxyfasudil (3 or 10 mg/kg, i.p.) once a day for 6 weeks. Wistar rats and SHRs treatment with vehicle were used as age-matched controls. Penile cGMP concentrations and Rho kinase activities were determined, and penile function was estimated by organ bath studies with norepinephrine-induced contractions and acetylcholine-induced relaxations. The participation mRNA levels of eNOS and participation protein levels of eNOS and phosphorylated eNOS were investigated by quantitative real-time PCR methods and immunoblot analysis, respectively. The SHR showed significantly decreased cGMP concentrations, increased Rho kinase activities, norepinephrine-induced hyper-contractions, and acetylcholine-induced hypo-relaxations in the penile tissue. Treatment with hydroxyfasudil significantly improved the decreased penile cGMP concentrations, the increased Rho kinase activities, the increased norepinephrine-induced contractions, and the decreased acetylcholine-induced relaxation in a dose-dependent manner. Although there were no significant differences in expression protein levels of eNOS among any of the groups, down-regulation of eNOS mRNAs as well as phosphorylated eNOS were significantly ameliorated after treatment with hydroxyfasudil. Our data suggest that hydroxyfasudil ameliorates hypertension-associated dysfunction of NO-induced relaxation in corpus cavernosum smooth muscle possibly via inhibition of the Rho-Rho kinase pathway and activation of NO-eNOS pathway in the SHR.

The role of K ATP channels on ischemia-reperfusion injury in the rat testis.

AIMS: To investigate the participation of K(ATP) channels on the ischemia-reperfusion (IR)-induced apoptosis in the rat testis. MAIN METHODS: Eight-week-old male Sprague-Dawley rats were divided into three groups: control and IR rats without or with cromakalim (300 µg/kg intraperitoneally), 30 min before the induction of ischemia. The right testicular artery and vein were clamped to induce ischemia in the testis. Sixty minutes after the ischemia, a 24h period of reperfusion followed. Then, expressions of K(IR)6.1, K(IR)6.2, caspase-3, PARP, Fas, FasL, and K(IR)6.1 and K(IR)6.2 mRNAs were investigated by Western blot analyses and real-time PCR methods, respectively. Furthermore, testicular tissues were processed for histological evaluation and TUNEL staining. KEY FINDINGS: Expressions of K(IR)6.1 protein and mRNA were more than 10-fold of those of K(IR)6.2 protein and mRNA in the testis. IR significantly increased the expressions of K(IR)6.1 protein and mRNA as well as K(IR)6.2 mRNA, caspase-3, and TUNEL index in the testis compared to the control. PARP expressions were significantly lower in the IR group than those of the control. Histologically, severe acute germ cell damage was observed in the IR testis. Treatment with cromakalim ameliorated these parameters compared to the non-treated IR group. There were no significant differences on Fas, FasL and protein level of K(IR)6.2 expressions between any of the groups. SIGNIFICANCE: Treatment with cromakalim has a protective effect against IR-induced testicular damage via activating K(ATP) channels. This is the first study to give evidence for the advantageous effect of cromakalim in the germ cell-specific apoptosis induced by testicular IR.

Nicorandil ameliorates hypertension-related bladder dysfunction in the rat.

AIMS: There is increasing evidence that ischemia is one of the main etiology in overactive bladder (OAB), and that nicorandil prevents OAB. We investigated the effect of nicorandil on hypertension-related bladder dysfunction in spontaneously hypertensive rats (SHRs). METHODS: Twelve-week-old SHRs received six-weeks treatment with nicorandil (0, 3, or 10 mg/kg, i.p. every day). Wistar rats were used for normotensive controls. Six weeks after nicorandil treatment, the bladder blood flow was estimated by hydrogen clearance method, and the bladder functions were estimated by voiding behavior studies and functional studies. Tissue levels of nerve growth factor (NGF) were measured by ELISA method. Furthermore, the participation levels of K(ATP) channel pores were investigated by real-time PCR. RESULTS: SHRs showed significant increases in blood pressure, micturition frequency, tissue levels of NGF and expressions of both K(IR) 6.1 and K(IR) 6.2 mRNAs, and a significant decrease in the bladder blood flow. The carbachol-induced contractile responses were similar in all groups. Although both doses of nicorandil failed to decrease the blood pressure, nicorandil significantly decreased the micturition frequency, tissue levels of NGF and increased the bladder blood flow in a dose dependent manner. The expressions of K(IR) 6.1 and K(IR) 6.2 mRNAs were slightly up-regulated by the low dose of nicorandil, whereas the high dose of nicorandil significantly up-regulated those expressions compared to non-treated SHRs. CONCLUSIONS: These data indicate that nicorandil prevents hypertension-related bladder dysfunction in the SHR, which may be related to its effect on the increased blood flow in the bladder.

Pharmacological preconditioning of ATP-sensitive potassium channel openers on acute urinary retention-induced bladder dysfunction in the rat.

UNLABELLED: What's known on the subject? and What does the study add? Acute urinary retention (AUR) and catheterization for AUR (AURC) or drainage of the urine is a well established cause of bladder dysfunction. Previously, we reported that the induction of AURC significantly reduced contractile responses to both carbachol and KCl compared with a control group, and that this reduction was prevented by nicorandil and cromakalim in a dose-dependent manner; however, although we reported a possible beneficial effect of nicorandil and cromakalim on bladder dysfunction caused by AURC, its molecular mechanism is still unknown. Our study establishes that nicorandil and cromakalim, but not glibenclamide, prevent AURC-induced bladder dysfunction via up-regulation of both K(IR)6.1 and K(IR)6.2 with a subsequent decrease in oxidative stress and decreased induction of apoptosis in the bladder. OBJECTIVE: To investigate whether ATP-sensitive potassium (K(ATP)) channel openers prevent bladder injury after acute urinary retention (AUR) and subsequent catheterization for AUR (AURC) in the rat. MATERIALS AND METHODS: Eight-week-old male Sprague-Dawley rats were divided into five groups: a sham-operated control group, an AUR group, and three AUR groups treated with: nicorandil (10 mg/kg); cromakalim (300 µg/kg); or glibenclamide (5 mg/kg). AUR was induced by intravesical infusion of 3.0 mL of saline via cystostomy with simultaneous clamping of the penile urethra and, after 30 min of AUR, the bladder was allowed to drain for 60 min. After the experimental period, bladder function was assessed using organ bath techniques (carbachol and KCl), and by measuring tissue levels of 8-isoprostane, a marker of oxidative stress. The participation levels of K(ATP) channel pores were investigated using ELISA and real-time PCR methods, respectively. The degree of apoptosis was estimated using the TUNEL method in the bladder smooth muscle and epithelium. RESULTS: The AURC group showed significantly decreased contractile responses to carbachol and KCl, and significant increases in tissue 8-isoprostane levels and apoptosis index in the epithelium compared with the control group. Nicorandil and cromakalim, but not glibenclamide, significantly prevented these AURC-induced alterations. The expressions of K(IR)6.1 and K(IR)6.2 mRNAs were significantly up-regulated by the induction of AURC. Nicorandil and cromakalim, but not glibenclamide, significantly up-regulated expressions of K(IR)6.1 and K(IR)6.2 mRNAs in the bladder compared with the AUR group. CONCLUSION: Our data indicate that nicorandil and cromakalim, but not glibenclamide, prevent AURC-induced bladder dysfunction via activation of K(ATP) channels, with a subsequent decrease in oxidative stress and decreased induction of apoptosis.

Strategies of newcastle disease vaccination for commercial ostrich farms in Japan.

Strategies of Newcastle disease (ND) vaccination were demonstrated in a commercial ostrich farm in Japan. Three of 13 seven-month-old ostriches kept in a pen were vaccinated with a live ND vaccine by eye dropping for the 1st and 2nd vaccinations and spraying for the 3rd to 5th vaccinations. Antibodies against ND virus (NDV) were detected in all of the unvaccinated ostriches by virus neutralization test. At 2.5 months post final vaccination, 2 ostriches introduced into the pen raised antibodies against NDV. These data indicate that NDV may be transmitted from vaccinated to unvaccinated ostriches in the flock and that the virus may be sustained for a certain period in the flock. These data may be helpful for ND vaccination management in ostrich farms.

Effect of silodosin on detrusor overactivity in the male spontaneously hypertensive rat.

UNLABELLED: Recently, several studies have suggested that detrusor overactivity (DO) is the result of bladder ischaemia. Hypertension could affect pelvic arterial blood flow, resulting in loss of smooth muscle in the bladder with resultant loss of bladder compliance. Spontaneously hypertensive rats are considered a valuable tool for exploring the pathogenesis of DO. Some reports indicate that α(1) adrenoceptor antagonists improve chronic ischaemia of the lower urinary tract in patients with LUTS, with concomitant improvement of their symptoms as well as improvement of DO through an increased bladder blood flow (BBF) in the rat with bladder outlet obstruction. However, the mechanism of improvement of silodosin on storage or irritative symptoms is not well investigated and is still unclear. Silodosin prevents hypertension-related DO in the SHR via several possible mechanisms. One possible mechanism of the efficacy of silodosin to the DO includes the improvement of the BBF. OBJECTIVE: To investigate the effect of the α(1A) selective adrenoceptor antagonist, silodosin, on hypertension-related detrusor overactivity (DO) and its possible mechanism in spontaneously hypertensive rats (SHRs). MATERIALS AND METHODS: Twelve-week-old male SHRs received treatment with silodosin (100 µg/kg perorally) once daily for 6 weeks; vehicle-treated Wistar rats and vehicle-treated SHRs were used for our study. Six weeks after silodosin treatment, voiding functions were estimated by voiding behaviour and cystometric studies in all groups. The bladder blood flow was measured by the hydrogen clearance method, and tissue levels of nerve growth factor (NGF) and calcitonin gene-related peptide (CGRP) were measured by enzyme-linked immunosorbent assay (ELISA). Furthermore, the expressions of α1 adrenoceptor subtype mRNAs in the bladder were investigated by real-time PCR method. RESULTS: The SHRs showed significant increases in blood pressure, micturition frequency and tissue levels of NGF and CGRP in the bladder. Moreover, the SHRs showed significant decreases in bladder blood flow and single voided volume estimated by both voiding behaviour and cystometric studies compared with those in the Wistar rats. Six weeks of treatment with silodosin significantly ameliorated hypertension-related alterations of these variables with concomitant small changes of blood pressure. The expression levels of α1 adrenoceptor subtype mRNAs in the bladder were similar in all the groups and the rank order was α1A = α1D > α1B in all groups. However, there were no significant differences in the expressions of α(1A) adrenoceptor mRNAs between any groups. CONCLUSION: The data in the present study suggest that silodosin normalizes hypertension-related DO in SHRs, which could be related to its effect on the increased blood flow in the bladder.

Rhos and Rho kinases in the rat prostate: their possible functional roles and distributions.

As there is increasing evidence that Rho-Rho kinase (ROCK) pathway plays an important role in the proliferation and contraction in many tissues, we investigated the contractile role of a ROCK inhibitor, fasudil, and the distribution of RhoA, RhoB, RhoC, ROCK1, and ROCK2 in the rat prostate. Twelve-week-old Sprague-Dawley rat prostate was used in this study. Rat prostatic contractile responses induced by carbachol and norepinephrine were investigated in organ bath studies without or with 10(-7), 10(-6), and 10(-5) M of a non-selective ROCK inhibitor, fasudil. Immunoblot analysis and immunohistochemical staining were performed to investigate the participation levels of RhoA, RhoB, RhoC, ROCK1, and ROCK2. The E(max) values induced by carbachol and norepinephrine were similar in the rat prostate. Fasudil significantly inhibited carbachol- or norepinephrine-induced prostatic contractions in a dose-dependent manner. Fasudil 10(-5) M reduced the initial prostatic contraction (without fasudil) to 56.7 ± 5.9% for carbachol and to 45.7 ± 12.3% for norepinephrine. Amounts of RhoA, RhoB, RhoC, ROCK1, and ROCK2 were detected by immunoblot analysis in the prostate. Immunohistochemical study revealed that RhoA, RhoB, RhoC, ROCK1, and ROCK2 were all positive in the prostatic smooth muscle, while there were some differences of distributions of Immunoreactivities between these enzymes in the prostatic glandula. Our data indicated that rat prostate contains RhoA, RhoB, RhoC, ROCK1, and ROCK2, which play an important role in the autonomic nerve-mediated contractile responses in the prostate.