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The objective of the present study is to develop a high time resolution ultrasonic velocity profiler (UVP) system by improving a signal processing and apply it to turbulent flow. The UVP system is able to obtain one-dimensional velocity distribution on an ultrasonic beam line from echo signals reflected at tracer particles. The position is determined from a time delay between emission of ultrasound pulse and reception of corresponding echo signals. The time resolution of conventional UVP systems is limited to the order of 10 ms at the best due to its signal processing technique, that needs more than a few ten echo signals to obtain the velocity distribution. In order to improve the time resolution of the UVP, we use a cross-correlation technique to estimate a time difference between two echo signals of a pair of emissions of ultrasound pulses. The pipe flow was examined by the present UVP system with the new signal processing. The result showed in a good agreement with that of laser doppler velocimetry measurement. In this experiment, we could improve the time resolution of UVP up to 500 μs to obtain a line profile of velocities and demonstrated a possibility to realize measurements of a smaller time scale velocity fluctuation in flows. [Copyright &y& Elsevier]

Bcl-2 family proteins regulate programmed cell death, and may play an important role in the selection of lymphocytes. We investigated the expression of Bcl-2, Bcl-x, Bax, Bak and Bim in human lymphocytes using flow-cytometry. Bcl-2 was down-regulated in CD4+8+ (DP) thymocytes and CD19+38+ tonsillar lymphocytes (GC B cells). Among DP thymocytes, cells co-expressing CD69 up-regulated Bcl-2, suggesting that the role of Bcl-2 is promoting survival of positively selected DP cells. Unexpectedly, the expression level of Bcl-x was higher in DP cells than in Single Positive (SP) cells and in CD69+ DP thymocytes it was lower than in CD69+ DP thymocytes. Expression of Bim was low in DP thymocytes but high in a subset of GC B cells. Bim and Bax were expressed more highly in SP than in DP thymocytes. Among peripheral blood lymphocytes (PBL), CD8+ T cells expressed a ∼ten-fold higher level of Bcl-x than CD4+ T cells while both subsets expressed similar levels of Bcl-2. Bak expression was low and Bim expression was absent in PBL. These results suggest that not only Bcl-2 but other members of the Bcl-2 family are involved in T cell development in the thymus and affinity maturation of B cells in the germinal center. [Copyright &y& Elsevier]

Discusses the effect of 17-beta-estradiol (E2) on the reproduction of Japanese medaka, Oryzias latipes. Concentrations of E2 in the flow-through systems; Total number of eggs and fertility during the exposure period; Mortality and abnormal appearance in adult medaka.

Survivin is a member of the inhibitor-of-apoptosis (IAP) family. It has been reported to be expressed during development, but not in differentiated normal tissue. However, its expression has been reported to be high in the thymus. To assess the role of survivin in human thymocyte development, we investigated the expression of survivin using reverse-transcriptase-polymerase chain reaction, flow cytometry, and immunohistochemistry in freshly isolated human thymocytes. Survivin was expressed in all thymocyte subsets but its expression level was developmentally regulated. Its expression was low in the double negative (DN) thymocytes, upregulated in double positive (DP) thymocytes, and was highest in the T-cell receptorhigh, late DP thymocytes; it was then downregulated in the single positive thymocytes and negative in the peripheral blood T cells. Moreover, there was a positive correlation between the expression of survivin and that of CD69 and Bcl-2 in DP thymocytes. These results suggest that survivin may play an important role in the T-cell development in the human thymus. [Copyright &y& Elsevier]

Immunocytochemistry was used to investigate the distribution of cells reacting with specific antibodies against glutathione S -transferase (GST) μ and π in rat circumvallate and foliate taste buds; the findings were confirmed by Western blotting. Double immunofluorescence staining for protein gene product (PGP) 9.5 and GST subunits allowed the classification of taste bud cells of both papillae into: (i) cells immunoreactive to either PGP 9.5 or GST subunit antibody; (ii) cells immunoreactive to both antibodies; and (iii) cells that did not react with either of these antibodies. Immunoelectron microscopy revealed that most GST subunit-immunoreactive cells seemed to be either type II or type III cells based on their ultrastructure. Since PGP 9.5 is now widely used as a marker for type III cells in mammalian taste buds, it seems reasonable to believe that most GST subunit-immunoreactive cells are type II cells. Whether cells immunoreactive for both PGP 9.5 and GST subunits consitute a small subpopulation of type III cells or whether they are intermediate forms between type II and III cells is under investigation. No type I cells reacted with antibodies against GST subunits in the present study. GST subunits in taste bud cells may participate in xenobiotic metabolism of certain substances exposed to taste pits, as already shown for olfactory epithelium. [ABSTRACT FROM AUTHOR]

Bilateral partial anomalous venous connections are rare. Here, we present a patient who underwent the modified Warden procedure to reroute the superior vena cava with the partial anomalous veins to the left atrium and reconstruct the innominate vein and superior vena cava with a polytetrafluoroethylene conduit to the right atrial appendage. The procedure was successfully performed without using foreign materials in the pulmonary venous route. Furthermore, it prevented the obstruction of blood flow., (Copyright © 2019 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

The tuberomammillary nucleus (TMN) of the posterior hypothalamus has a high density of histaminergic neurons, the projection fibers of which are present in many areas of the brain, including the nucleus tractus solitarius (NTS), which controls arterial pressure (AP). In this study, we investigated whether the TMN-NTS pathway is involved in central cardiovascular regulation. Bicuculline, a gamma-aminobutyric acid type A (GABA A ) receptor antagonist, was microinjected into the ventral TMN of anesthetized rats and its effects on AP and heart rate (HR) were observed. We also evaluated the effect of cetirizine, an H 1 receptor antagonist, microinjected into the NTS on cardiovascular responses induced by electrical stimulation of the TMN Both AP and HR increased following bicuculline microinjection into the ventral TMN Similar pressor and tachycardic responses were observed after electrical stimulation of the ventral TMN Microinjection of cetirizine into the NTS partially inhibited the pressor response but had no effect on HR Finally, the treadmill test was associated with a high level of c-Fos expression in both ventral TMN and NTS neurons. These results suggest that the TMN-NTS pathway is involved in regulation of AP, presumably under a high-arousal phase, such as that during exercise., (© 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

The effects of adding desipramine-containing fluorescence polymer (poly(10,11-dihydro-5-[3-(N-methylamino)propyl]dibenz[b,f]azepine-2,8-diyl-alt-9,9-didodecylfluorene-2,7-diyl, PAzep-Fl) to resins were investigated. When composites were prepared by a reactive blend of poly(lactic acid) (PLA), PAzep-Fl, and diisocyanate, the molecular weight of the obtained composite was larger than that of the composite, which was blended without PAzep-Fl; this suggested that chemical bonding occured between PLA and PAzep-Fl via diisocyanate. The effects of adding PAzep-Fl in two kinds of resins/lysine triisocyanate (LTI) blend were also investigated. The addition of LTI to resins/PAzep-Fl blend composites afforded a shorter wavelength shift of the fluorescence λ max due to the release of the aggregation of PAzep-Fl by LTI. This suggested that the compatibility of two kinds of resins could be estimated by this simple method. The addition of PAzep-Fl to PLA/poly(ε-caprolactone) (PCL)/LTI blends improved the impact strength because of chemical bond formation between PAzep-Fl and the resins (PLA and PCL) via LTI.

Both the Damus-Kaye-Stansel procedure and pulmonary artery banding provide an effective palliative technique in certain subsets of patients, but this combination of procedures is potentially hazardous in pulmonary valve geometry. This is a matter of vital concern, especially in patients whose pulmonary arteries have to work after operations as the major systemic output routes. In this report, we present a novel surgical modification that builds a systemic output route without causing valve deformities., (Copyright © 2015 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Objective: Elderly hypertensive patients are characterized by blood pressure (BP) variability, impaired autonomic function, and vascular endothelial dysfunction and stiffness. However, the mechanisms causing these conditions are unclear. The present study examined the effect of angiotensin receptor blockers (ARBs) on aged spontaneously hypertensive rats (SHR)., Methods: We surgically implanted telemetry devices in SHR and WKY at the age of 15 weeks (Young) and 80 weeks (Aged). Aged SHR were orally administered either olmesartan or valsartan once daily at 19:00 h (at the beginning of the dark period (active phase)) for 4 weeks to examine the effects on BP variability, impaired autonomic function, and vascular senescence., Results: Aging and hypertension in SHR additively caused the following: increased low frequency (LF) power of systolic BP, a decreased spontaneous baroreceptor reflex gain (sBRG), increased BP variability, increased urinary norepinephrine excretion, increased vascular senescence-related beta-galactosidase positive cells and oxidative stress. Treatment with olmesartan or valsartan significantly ameliorated these changes in aged SHR. However, olmesartan ameliorated these changes in aged SHR better than valsartan. The reductions in BP caused by olmesartan in aged SHR were sustained longer than reductions by valsartan. This result indicates longer-lasting inhibition of the AT1 receptor by olmesartan than by valsartan., Conclusion: ARBs ameliorated autonomic dysfunction, BP variability, and vascular senescence in aged SHR. Olmesartan ameliorated the aging-related disorders better than valsartan and was associated with longer-lasting AT1 receptor inhibition by olmesartan. Thus, the magnitude of improvement of these aging-related abnormalities differs for ARBs., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Background: Although renal denervation (RD) is shown to reduce blood pressure significantly in patients with resistant hypertension, the benefit of RD in prevention of stroke is unknown. We hypothesized that RD can prevent the incidence of stroke and brain injury in hypertensive rats beyond blood pressure lowering., Methods and Results: High-salt-loaded, stroke-prone, spontaneously hypertensive rats (SHRSP) were divided into 4 groups: (1) control; (2) sham operation; (3) bilateral RD; and (4) hydralazine administration to examine the effect of RD on stroke and brain injury of SHRSP. RD significantly reduced the onset of neurological deficit and death in SHRSP, and this protection against stroke by RD was associated with the increase in cerebral blood flow (CBF), the suppression of blood-brain barrier disruption, the limitation of white matter (WM) lesions, and the attenuation of macrophage infiltration and activated microglia. Furthermore, RD significantly attenuated brain oxidative stress, and NADPH oxidase subunits, P67 and Rac1 in SHRSP. On the other hand, hydralazine, with similar blood pressure lowering to RD, did not significantly suppress the onset of stroke and brain injury in SHRSP. Furthermore, RD prevented cardiac remodeling and vascular endothelial impairment in SHRSP., Conclusions: Our present work provided the first experimental evidence that RD can prevent hypertensive stroke and brain injury, beyond blood pressure lowering, thereby highlighting RD as a promising therapeutic strategy for stroke as well as hypertension.

Background: Although renal denervation significantly reduces blood pressure in patients with resistant hypertension, the role of the renal nerve in hypertension with metabolic syndrome is unknown. We investigated the impact of long-term renal denervation on SHR/NDmcr-cp(+/+) (SHRcp) rats, a useful rat model of metabolic syndrome, to determine the role of the renal nerve in hypertension with metabolic syndrome., Methods and Results: SHRcp rats were divided into (1) a renal denervation (RD) group and (2) a sham operation group (control) to examine the effects of long-term RD on blood pressure circadian rhythm, renal sodium retention-related molecules, the renin-angiotensin-aldosterone system, metabolic disorders, and organ injury. RD in SHRcp rats not only significantly reduced blood pressure but also normalized blood pressure circadian rhythm from the nondipper to the dipper type, and this improvement was associated with an increase in urinary sodium excretion and the suppression of renal Na(+)-Cl(-) cotransporter upregulation. RD significantly reduced plasma renin activity. RD significantly prevented cardiovascular remodeling and impairment of vascular endothelial function and attenuated cardiovascular oxidative stress. However, RD failed to ameliorate obesity, metabolic disorders, and renal injury and failed to reduce systemic sympathetic activity in SHRcp rats., Conclusions: By including the upregulation of the Na(+)-Cl(-) cotransporter, the renal sympathetic nerve is involved in the disruption of blood pressure circadian rhythm as well as hypertension in metabolic syndrome. Thus, RD seems to be a useful therapeutic strategy for hypertension with metabolic syndrome.

Background: This study was performed to determine the characteristics and mechanism of hypertension in SHR/NDmcr-cp(+/+) rats (SHRcp), a new model of metabolic syndrome, with a focus on the autonomic nervous system, aldosterone, and angiotensin II., Methods and Results: We measured arterial blood pressure (BP) in SHRcp by radiotelemetry combined with spectral analysis using a fast Fourier transformation algorithm and examined the effect of azilsartan, an AT1 receptor blocker. Compared with control Wistar-Kyoto rats (WKY) and SHR, SHRcp exhibited a nondipper-type hypertension and displayed increased urinary norepinephrine excretion and increased urinary and plasma aldosterone levels. Compared with WKY and SHR, SHRcp were characterized by an increase in the low-frequency power (LF) of systolic BP and a decrease in spontaneous baroreflex gain (sBRG), indicating autonomic dysfunction. Thus, SHRcp are regarded as a useful model of human hypertension with metabolic syndrome. Oral administration of azilsartan once daily persistently lowered BP during the light period (inactive phase) and the dark period (active phase) in SHRcp more than in WKY and SHR. Thus, angiotensin II seems to be involved in the mechanism of disrupted diurnal BP rhythm in SHRcp. Azilsartan significantly reduced urinary norepinephrine and aldosterone excretion and significantly increased urinary sodium excretion in SHRcp. Furthermore, azilsartan significantly reduced LF of systolic BP and significantly increased sBRG in SHRcp., Conclusions: These results strongly suggest that impairment of autonomic function and increased aldosterone in SHRcp mediate the effect of angiotensin II on circadian blood pressure rhythms.

Background: The Fontan operation for patients with one available lung is an extremely challenging situation. However, few reports are available on this procedure. The purpose of this study was to describe outcomes of one-lung Fontan operation., Methods: A retrospective multicenter study was performed. Twelve of 1,142 patients whose data were recorded here underwent one-lung Fontan operation between September 1989 and October 2009. Preoperative, operative, and postoperative data were reviewed., Results: Median age at operation was 3.5 years (range, 1.0 to 22.8), the preoperative mean pulmonary pressure was 11.5±3.3 mm Hg (range, 7.0 to 18.0), the ventricular ejection fraction was 58%±13% (range, 39 to 76), and end-diastolic ventricular pressure was 7.5±3.5 mm Hg (range, 1.0 to 12.0). The available lung was right in 9 patients and left in 3 patients. Eleven patients underwent a two-staged Fontan completion. Extracardiac conduit total cavopulmonary connection, intraatrial extracardiac conduit total cavopulmonary connection, and atriopulmonary connection were performed in 10 patients, 1 patient, and 1 patient, respectively. The estimated actuarial survival was 83% at 1 year, 73% at 5 years, and 73% at 10 years. Impaired ventricular function was found to be a significant risk factor for mortality by univariate analysis (43.0%±9.5% versus 64.0%±9.5%, p<0.01), but not by multivariate analysis., Conclusions: One-lung Fontan operation can be performed with an acceptable midterm to long-term mortality rate in patients without impaired ventricular function. Thus, absence of one lung itself is not a contraindication to the Fontan operation., (Copyright © 2012 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Understanding how the 24-hour blood-pressure rhythm is programmed has been one of the most challenging questions in cardiovascular research. The 24-hour blood-pressure rhythm is primarily driven by the circadian clock system, in which the master circadian pacemaker within the suprachiasmatic nuclei of the hypothalamus is first entrained to the light/dark cycle and then transmits synchronizing signals to the peripheral clocks common to most tissues, including the heart and blood vessels. However, the circadian system is more complex than this basic hierarchical structure, as indicated by the discovery that peripheral clocks are either influenced to some degree or fully driven by temporal changes in energy homeostasis, independent of the light entrainment pathway. Through various comparative genomic approaches and through studies exploiting mouse genetics and transgenics, we now appreciate that cardiovascular tissues possess a large number of metabolic genes whose expression cycle and reciprocally affect the transcriptional control of major circadian clock genes. These findings indicate that metabolic cycles can directly or indirectly affect the diurnal rhythm of cardiovascular function. Here, we discuss a framework for understanding how the 24-hour blood-pressure rhythm is driven by the circadian system that integrates cardiovascular and metabolic function.

Objectives: The brainstem nucleus of the solitary tract (nucleus tractus solitarii, NTS) is a pivotal region for regulating the set-point of arterial pressure, the mechanisms of which are not fully understood. Based on evidence that the NTS exhibits O2-sensing mechanisms, we examined whether a localized disturbance of blood supply, resulting in hypoxia in the NTS, would lead to an acute increase in arterial pressure., Methods: Male Wistar rats were used. Cardiovascular parameters were measured before and after specific branches of superficial dorsal medullary veins were occluded; we assumed these were drainage vessels from the NTS and would produce stagnant hypoxia. Hypoxyprobe-1, a marker for detecting cellular hypoxia in the post-mortem tissue, was used to reveal whether vessel occlusion induced hypoxia within the NTS., Results: Following vessel occlusion, blood flow in the dorsal surface of the medulla oblongata including the NTS region showed an approximately 60% decrease and was associated with hypoxia in neurons located predominantly in the caudal part of the NTS as revealed using hypoxyprobe-1. Arterial pressure increased and this response was pronounced significantly in both magnitude and duration when baroreceptor reflex afferents were sectioned., Conclusion: These results suggest that localized hypoxia in the NTS increases arterial pressure. We suggest this represents a protective mechanism whereby the elevated systemic pressure is a compensatory mechanism to enhance cerebral perfusion. Whether this physiological mechanism has any relevance to neurogenic hypertension is discussed.

Objectives: Recent studies have demonstrated that pro-inflammatory molecules such as junctional adhesion molecules-1 are highly expressed in the nucleus tractus solitarii (NTS) of the spontaneously hypertensive rat (SHR), compared to normotensive rats (Wistar-Kyoto rats: WKY), suggesting that the NTS of SHR may exhibit an abnormal inflammatory state. In the present study, we tested whether gene expression of inflammatory markers such as cytokines and chemokines is altered in the NTS of SHR and whether this contributes to the hypertensive phenotype in the SHR., Methods: We have performed RT Profiler PCR arrays in the NTS of SHR and WKY, which were designed to specifically target major cytokines/chemokines and their receptors. To validate PCR array results quantitative RT-PCR was performed. Microinjection studies using anesthetized rats were also carried out to examine whether validated inflammatory molecules exhibit functional roles on cardiovascular regulation at the level of the NTS., Results: Five inter-related transcripts were identified to be differentially expressed between the NTS of SHR and WKY. They include chemokine (C-C motif) ligand 5 (Ccl5), and its receptors, chemokine (C-C motif) receptor 1 and 3. All of them were down-regulated in the NTS of SHR compared to WKY. Moreover, we found that the protein Ccl5 microinjected into the NTS significantly decreased baseline arterial pressure and that the response was greater in the SHR compared to the WKY (-33.2±3.2 vs. -8.8±1.6 mmHg, P<0.001), demonstrating that its down-regulation in the NTS may contribute to hypertension in the SHR., Conclusion: We suggest that gene expression of specific chemokines may be down-regulated to protect further inflammatory reactions in the NTS of SHR at the expense of arterial hypertension.

Background: Sema4D/CD100 is a type of class 4 semaphorin, exhibiting crucial roles in growth cone guidance in developing neurons. Sema4D is widely expressed throughout the central nervous system in embryonic mouse brain, and is selectively localized to oligodendrocytes and myelin in the postnatal brain. However, direct evidence of the actual involvement of Sema4D in the neuronal network development crucial for neurobehavioral performance is still lacking. The present study therefore examined whether Sema4D deficiency leads to abnormal behavioral development., Methods: Both wild-type and Sema4D-deficient mice were subjected to behavioral analyses including open-field, adhesive tape removal, rotarod tests and a water maze task., Results: Open-field tests revealed increased locomotor activity in Sema4D-deficient mice with less percentage of time spent in the center of the field. In both the adhesive tape removal and rotarod tests, which examine motor coordination and balance, Sema4D-deficient mice showed significantly superior performance, suggesting facilitated motor behavior. Both Sema4D-deficient and wild-type mice successfully learnt the water maze task, locating a hidden escape platform, and also showed precise memory for the platform position in probe tests. However, the swimming speed of Sema4D-deficient mice was significantly faster than that of wild-type mice, providing further evidence of their accelerated motor behavior., Conclusion: Our mouse behavioral analyses revealed enhanced motor activity in Sema4D-deficient mice, suggesting the crucial involvement of Sema4D in the neurodevelopmental processes of the central structures mediating motor behavior in mice.

Objectives: Despite pacemaker therapy in children and adolescents favoring an initial epicardial approach, predictors of lead failure have not been well clarified. The aim of this study was to assess the long-term outcomes and to determine predictors affecting lead durability in pediatric pacing therapy., Methods: We reviewed the outcomes of 109 consecutive pacing leads implanted in 55 patients (median age, 5.2 years; range, 31 days-15.8 years), including 38 atrial and 71 ventricular leads. They consisted of 58 (53%) fishhooks, 37 (34%) screw-in leads, and 14 (13%) steroid-eluting suture-on leads. Seventy (64%) were implanted in patients with structural heart disease., Results: The leads were followed for a median of 6.4 years (range, 3 days-22.9 years). Lead failure occurred in 29 leads (27%; median of 8.4 years after implantation). Exit block or elevation of pacing threshold was the most common cause (n = 18), but failures did not directly cause patient death. The overall 1-, 5-, 10-, and 15-year lead survivals were 100%, 89.0%, 72.5%, and 55.5%, respectively. Multivariate Cox analysis revealed concurrent structural heart disease (relative risk, 2.85; 95% confidence interval, 1.27-6.42; P = .011) to be the only significant predictor of lead failure., Conclusions: Epicardial leads provide a reliable technique for managing rhythmic disturbance problems in the pediatric population. The only significant predictor of lead failure is the presence of structural heart disease.

The subacute toxicity of toluene-2,4-diamine (2,4-TDA) on marine fish was investigated in laboratory toxicity tests using red sea bream, Pagrus major. The fish were exposed to subacute concentrations of 2,4-TDA (measured concentrations: 0.0628, 0.234 and 0.878 mg/L) during the 14-day toxicity test and the effects on growth as well as on some biochemical parameters in the plasma, gills, liver and kidney structures were studied. The body weight and body length of the red sea bream exposed to the highest concentration were lower than those of the control. This study also showed a tendency towards decreases in three hematological parameters, i.e., the number of red blood cells, hemoglobin level and hematocrit, as well as blood chemical parameters in the plasma, i.e., the levels of total cholesterol, triglyceride, total protein and albumin, of fish exposed to the high concentrations. These findings were attributable to the reduced feeding of fish exposed to this concentration. In addition, cytoplasmic changes were observed in the cells of fish exposed to the highest concentration. It is suggested that the changes were caused by the decrease in feed efficiency derived from the exposure to 2,4-TDA.

Background: Down syndrome patients are characterized by early progression of pulmonary vascular obstructive disease because of insufficient thickness of the pulmonary arterial media. For those with congenital heart disease (CHD) associated with pulmonary hypertension (PH), a 2-stage procedure of pulmonary artery banding (PAB) and then intracardiac repair (ICR) in early infancy is performed to prevent such pulmonary vascular diseases in early infancy., Methods and Results: The subjects were 16 patients with Down syndrome who underwent lung biopsy during PAB and ICR. PAB was planned to be performed in early infancy and ICR approximately 1 year later. Efficacy of the 2-stage procedure was retrospectively examined with reference to pulmonary vascular disease and pulmonary diseases. The index of pulmonary vascular disease at PAB fulfilled the indication for ICR, and it was significantly lower at ICR than at PAB (p=0.0469); furthermore, PAB prevented progression of pulmonary diseases., Conclusions: The results of the lung biopsies support the safety of the 2-stage procedure and show that it is effective for Down syndrome patients with CHD and PH.

Death-associated protein kinase (DAPK) is a Ca2+/calmodulin-dependent serine/threonine kinase that is thought to mediate apoptosis. DAPK is highly expressed in hippocampal neurons which are essential elements for memory formation. To examine if DAPK is implicated in spatial learning and memory, both wild-type and DAPK-mutant mice were subjected to Morris water maze tests. DAPK-mutant mice were generated by deleting 74 amino acids from the catalytic kinase domain of DAPK, and were used to investigate roles of the DAPK kinase domain in regulating spatial memory. Both mutant and wild-type mice were able to learn the water maze tasks to locate a hidden escape platform. In the first probe test, mutant mice showed a more precise memory for platform position compared to wild-type mice. In the reversal training in which the platform was located opposite from the original position, DAPK-mutant mice exhibited superior spatial learning compared to wild-type mice. DAPK-mutant mice also showed a more precise memory than their wild-type littermates in the probe trial of reversal test. Thus, the present results revealed crucial implications of DAPK in regulating spatial memory in mice.

To determine the role of STAT6 transcription factors in brain function, we performed a battery of mouse behavioral analyses of STAT6-deficient mice. We recently showed that STAT6-deficient mice displayed increased locomotor activities and had reduced levels of dopamine transporter (DAT) expression in the striatum. To further examine the behavioral effects of STAT6 deficiency, we subjected STAT6-deficient mice to behavior task testing prepulse inhibition (PPI) of the auditory startle response. In acoustic prepulse conditions, STAT6-deficient mice displayed significantly lower levels of PPI in acoustic startle than did wild-type mice, indicating their sensorimotor gating deficits. Thus, STAT6 transcription factors may be crucially implicated in sensorimotor gating by modulating the expression of several genes, including DAT in brain neurons.

Death-associated protein kinase (DAPK) is a Ca(2+)/calmodulin-dependent serine/threonine kinase that is thought to mediate apoptosis. We previously showed that the kinase domain of DAPK is crucial for the induction of renal tubular cell apoptosis in chronic obstructive uropathy (COU) caused by a unilateral ureteral ligation. Here, we used DAPK-mutant mice, generated by the deletion of 74 amino acids from the catalytic kinase domain, to investigate the role of the DAPK kinase domain in regulating the p53 level following COU. The p53 expression levels in obstructed kidneys of wild-type and mutant mice were determined during the course of COU. Western blot analysis revealed that the p53 protein levels were significantly increased at 5 days after a ureteral ligation. This increase in the p53 level was significantly attenuated in mutant kidneys compared to wild-type kidneys. The obstructed kidneys of DAPK-mutant mice showed a significantly lower number of p53-expressing renal tubule cells than wild-type mice. These results are consistent with the hypothesis that DAPK stabilizes p53 protein in response to apoptosis-inducing stimuli. Thus, the present results suggest that the DAPK kinase domain is crucial for stabilizing p53 protein in renal tubular cell apoptosis in a mouse model of COU.

Semaphorins are a family of secreted and membrane-bound proteins, known to control axonal pathfinding. It was recently demonstrated that Semaphorin 4A (Sema4A) is crucially involved in immune cell activation. However, the role of Sema4A in the nervous system has not yet been clarified. To examine if Sema4A can function as a chemo-repulsive cue to growth cones of developing hippocampal neurons, a growth cone collapse assay with recombinant Sema4A was performed in primary hippocampal neurons cultured from E17 mice. In these primary hippocampal neurons, Sema4A induced a significant growth cone collapse as compared with the culture without Sema4A. The Sema4A-induced growth cone collapse could be blocked by Y-27632, a Rho-kinase inhibitor. Furthermore, immunocytochemical analysis with antibodies against Sema4A demonstrated the binding of recombinant Sema4A to the growth cones of hippocampal neurons. Thus, our data indicated that Sema4A could function as a chemo-repulsive cue by activating a receptor whose signal is transmitted to Rho-kinase and induced growth cone collapse of hippocampal neurons.

To determine the role of STAT6 transcription factors in brain function, we performed a battery of mouse behavioral analyses of STAT6-deficient mice and made comparisons with wild-type mice. STAT6-deficient mice were significantly more hyperactive in the final two blocks of an open field test, indicating abnormal habituation in completing the task. Two learning tasks, a water maze and a passive avoidance test, were mastered by STAT6-deficient mice as effectively as the wild-type. RT-PCR analysis suggested that the levels of dopamine transporter (DAT) mRNA may be lower in the midbrain of the mutant. In agreement with the finding, subsequent Western blotting and immunohistochemical analysis demonstrated lower levels of DAT protein in the mutant striatum. These results lead to the proposal that, in addition to being a pivotal transcription factor in the immune system, STAT6 may be crucially implicated in mouse behavior by modulating the expression of neuronal genes such as DAT.

Ghrelin is a gut-brain peptide and its endocrine activities are mediated by GH secretagogue receptor (GHSR)-1a. Des-acyl ghrelin does not activate GHSR-1a and is devoid of endocrine activities. While the microinjection of ghrelin into rat nucleus tractus solitarii (NTS) elicited hypotensive effects, this was not the case upon injection into GHSR-expressing rostral ventrolateral medulla or caudal ventrolateral medulla. To make clear the reason of the discrepancy between receptor distribution and neuronal responses, we examined the cardiovascular response of rats microinjected with des-acyl ghrelin into NTS. Intra-NTS injection of des-acyl ghrelin significantly reduced mean arterial pressure and heart rate. The hypotensive and bradycardic activity evoked by des-acyl ghrelin was not significantly different from that of native ghrelin. These results suggest that des-acyl ghrelin contribute to the regulation of cardiovascular control and that a receptor other than GHSR-1a exists in NTS.

To elucidate the contribution of signal transducer and activator of transcription (STAT) 6 to the pathophysiology of chronic renal injury, STAT6-/- mice were subjected to unilateral ureteral ligation together with wild-type control mice. STAT6-/- kidneys had more apoptotic cells and a greater influx of F4/80-positive cells than wild-type kidneys following ureteral obstruction. There was a much larger alpha-smooth muscle actin-positive area in STAT6-/- kidneys than in wild-type kidneys after ureteral ligation. However, renal fibrosis, as quantified by Masson-Trichrome staining, was not significantly exaggerated in STAT6-/- kidneys compared with wild-type kidneys. The accumulation of collagen I was significantly less in STAT6-/- kidneys than in wild-type kidneys. These observations indicate that the STAT6 signal transduction pathway exerts a protective role on renal cell apoptosis in chronic obstructive uropathy. Our findings also suggest that the STAT6 pathway may have a promotive effect on renal fibrosis by activating collagen synthesis following ureteral obstruction.

Death-associated protein kinase (DAPK) is a Ca2+/calmodulin-dependent serine/threonine kinase that is thought to mediate apoptosis. We have shown that the kinase domain of DAPK is crucial for the induction of renal tubular cell apoptosis in chronic obstructive uropathy (COU) created by unilateral ureteral ligation. DAPK-mutant mice, generated by deletion of 74 amino acids from the catalytic kinase domain, were used to investigate the role of the DAPK kinase domain in renal fibrosis following COU. Interstitial collagen and alpha-smooth muscle actin (alpha-SMA) expressions in situ were compared between obstructed kidneys in wild-type and mutant mice. As a result, tubulointerstitial fibrosis, as quantified by interstitial collagen expression, was significantly augmented in mutant kidneys compared with wild-type kidneys following COU. Furthermore, deletion of the kinase domain from DAPK significantly increased the appearance of alpha-SMA-positive myofibroblasts in the renal interstitium during COU. Thus, our results suggest that the kinase domain deleted by gene targeting plays a suppressive role for the development of renal fibrosis through inhibition of the tubular epithelial-to-mesenchymal transition in a mouse model of COU.

Death-associated protein kinase (DAPK) is a calcium/calmodulin-dependent serine/threonine kinase localized to renal tubular epithelial cells. To elucidate the contribution of DAPK activity to apoptosis in renal ischemia-reperfusion (IR) injury, wild-type (WT) mice and DAPK-mutant mice, which express a DAPK deletion mutant that lacks a portion of the kinase domain, were subjected to renal pedicle clamping and reperfusion. After IR, DAPK activity was elevated in WT kidneys but not in mutant kidneys (1785.7 +/- 54.1 pmol/min/mg versus 160.7 +/- 60.6 pmol/min/mg). Furthermore, there were more TUNEL-positive nuclei and activated caspase 3-positive cells in WT kidneys than in mutant kidneys after IR (24.0 +/- 5.9 nuclei or 9.4 +/- 0.6 cells per high-power field [HPF] versus 6.3 +/- 2.2 nuclei or 4.4 +/- 0.7 cells/HPF at 40 h after ischemia). In addition, the increase in p53-positive tubule cells after IR was greater in WT kidney than in mutant kidneys (9.9 +/- 1.4 cells/HPF versus 0.8 +/- 0.4 cells/HPF), which is consistent with the theory that DAPK activity stabilizes p53 protein. Finally, serum creatinine levels after IR were higher in WT mice than in mutant mice (2.54 +/- 0.34 mg/dl versus 0.87 +/- 0.24 mg/dl at 40 h after ischemia). Thus, these results indicate that deletion of the kinase domain from DAPK molecule can attenuate tubular cell apoptosis and renal dysfunction after IR injury.

Death-associated protein kinase (DAPK) is a Ca2+/calmodulin-dependent serine/threonine kinase that has been implicated as a positive mediator of apoptosis. However, little is known about the involvement of DAPK in the apoptosis associated with several pathological states, except for cancer. Here, DAPK-mutant mice were used in order to examine the role of DAPK in renal cell apoptosis in chronic obstructive uropathy (COU) created by unilateral ureteral ligation. These mice express mutant DAPK with a deletion of 74 amino acids from the catalytic kinase domain. Obstructed kidneys in wild-type and mutant mice were examined for both DAPK protein levels and renal cell apoptosis during the course of COU. Obstructed kidneys in wild-type and mutant mice showed a marked increase in the DAPK and mutant DAPK protein levels, respectively, at day 14 after ureteric ligation. The obstructed kidneys in DAPK-mutant mice displayed a significant attenuation of tubular cell apoptosis, compared with wild-type mice. In contrast, no significant difference in interstitial cell apoptosis was observed between the obstructed kidneys from wild-type and mutant mice. Thus, these results indicate that the part of the kinase domain deleted by the gene targeting is crucial for the execution of tubular cell apoptosis, but is not essential for interstitial cell apoptosis in a COU model in mice.

The expression of the death-associated protein kinase (DAPK) protein and promoter methylation in 11 human uterine and ovarian carcinoma cell lines originally established from histopathologically-different carcinoma tissues were examined to investigate the relationship between DAPK and carcinogenesis in female reproductive tissues. The 11 cell lines included three cervical carcinomas, three endometrial carcinomas and five ovarian carcinomas. Western blot analysis showed no detectable expression of DAPK protein in 4 cell lines (ME180, HOKUG, MCAS and OVK-18) while moderate levels of DAPK protein were readily detected in normal human endometrium, and normal murine uterus and ovary. Methylation-specific PCR of the 11 cell lines revealed that 5 carcinoma cell lines (ME180, HOKUG, MCAS, OVK-18 and HEC-1) expressed hypermethylated promoters in the DAPK genes, while DAPK promoters in the other 6 carcinoma cell lines remained unmethylated. These results indicate that DAPK protein expression is reduced or silenced in some human uterine and ovarian carcinoma cells by methylation of the DAPK gene promoter region. Therefore, reduced DAPK expression and methylation of the DAPK promoter may be involved in carcinogenesis of human uterine and ovarian tissues.

Background: Death-associated protein kinase (DAP kinase) is a Ca2+/calmodulin-dependent serine/threonine kinase implicated as a positive apoptosis mediator. However, little is known about DAP kinase involvement with apoptosis in renal diseases., Methods: In order to determine whether DAP kinase has a role in renal cell apoptosis in kidney diseases, we performed an immunohistochemical study using a monoclonal antibody to DAP kinase. Firstly, by examining the cellular distribution of DAP kinase in normal human renal tissues and cultured proximal tubule cells. We then used western blotting and immunohistochemical analysis to examine directly whether DAP kinase protein levels could be modulated in rat kidneys with chronic obstructive uropathy created by unilateral ureteric ligation., Results: Immunohistochemistry of normal human kidney tissues showed that DAP kinase was exclusively localized in the cytoplasm of renal tubule cells. Expression analysis of DAP kinase using cultured cells confirmed DAP kinase mRNA and protein presence in human renal tubule cells. Immunocytochemical analysis directly visualized DAP kinase in the cytoplasm of the renal tubule cells in culture. Finally, DAP kinase was found up-regulated in renal tubule cells of rat kidneys with chronic obstructive uropathy., Conclusions: Our study demonstrates that DAP kinase is localized to renal tubule cells, implying a crucial role for DAP kinase in renal tubular cell apoptosis in progressive renal diseases.

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of the egg yolk precursor vitellogenin (VTG) in the liver of medaka (Oryzias latipes) and was employed to establish an in vivo testing system for estrogen and estrogenic compounds using liver homogenates. Results of 3-month-old fish exposed to three reference chemicals (ethynylestradiol, methyltestosterone and flutamide) recommended by Organisation for Economic Co-operation and Development (OECD) for the validation showed the induction and decrease of vitellogenic responses, making the assay using the liver VTG of medaka a possible screening method for not only estrogens but also androgens. In addition, 21-day exposure of male fish to 4-tert-octylphenol and 4-nonylphenol produced concentration-dependent inductions of liver vitellogenin, with the lowest observed effect concentrations of 64.1 microg/L and 22.5 microg/L, respectively, while no significant VTG responses were observed in male and female fish exposed to tributyltin chloride and dibutyl phthalate. This study demonstrates that the VTG assay using liver homogenates from small fish such as medaka can be used as a screening method for environmental estrogens. This is because the sensitivity of the VTG response in medaka may be almost the same as that of other fish using blood samples.

Neuromedin U (NMU) is a brain-gut peptide with potent contractile effects on the uterus and smooth muscle. Intracerebroventricular injection of NMU reportedly decreased food intake and body-weight gain in the rat. We evaluated the effects of NMU delivered by microinjection into the rat nucleus tractus solitarius (NTS) on cardiovascular responses. At the concentrations used (5, 10 or 50 pmol), the intra-NTS injection of NMU in artificial cerebrospinal fluid produced a significant reduction in both the mean arterial pressure and heart rate. The hypotensive responses were dose dependent. Our findings suggest that NMU may act as a neurotransmitter or neuromodulative substance that causes excitation of neurons in the NTS and that it may play a role in cardiovascular regulation in vivo.

We exposed mature Japanese medaka (Oryzias latipes) to 4-nonylphenol (4-NP) to elucidate its reproductive effects and estrogenic responses. For each treatment, eight breeding pairs were exposed for 3 weeks at measured 4-NP concentrations of 24.8, 50.9, 101, and 184 micrograms/L. Egg production of the pairs during the third week of exposure (15-21 d) was significantly decreased in the > or = 101-microgram/L treatment group, and fertility was significantly decreased in the 184-microgram/L group. Induction of testis-ova was observed in male fish at all 4-NP concentrations, whereas abnormality of spermatogenesis was found only in those receiving the 184-microgram/L treatment. The gonadosomatic index of the males in the 184-microgram/L group was also remarkably reduced. Hepatic vitellogenin (VTG) levels were significantly increased in both sexes receiving 4-NP treatments of > or = 50.9 micrograms/L. Overall, the lowest-observed effective concentrations (LOECs) of 4-NP for decreasing reproductive capacity (fecundity and fertility) and eliciting estrogenic responses (induction of testis-ova and VTG) were 101 and < or = 24.8 micrograms/L, respectively. The LOEC for reduced reproduction in medaka was at least four-fold greater than that required to elicit estogenic responses in male fish. Thus, we suggest that the estrogenic responses expressed in male fish are not responsible for the reduction in reproductive capacity.

Objective: We assessed the current role of preoperative and intraoperative autologous blood donation in pediatric open-heart surgery., Methods: Group 1 consisted of 51 patients between 5 and 10 years old who underwent preoperative autologous blood donation. Group 2 consisted of 50 age-matched patients without preoperative donation as controls. Intraoperative donation was conducted in both groups prior to cardiopulmonary bypass. We evaluated perioperative blood cell count, blood loss, and the need for homologous blood products., Results: No serious complications occurred in preoperative or intraoperative donation. Total preoperative donation storage was 17.5 +/- 3.4 mL/kg. Intraoperative donation was 21.7 +/- 6.1 mL/kg in Group 1 and 12.8 +/- 4.0 mL/kg in Group 2 (p < 0.001). On admission, serum hemoglobin was lower in Group 1 (12.2 +/- 1.0 g/dL versus 13.6 +/- 1.6 g/dL, p < 0.001) but returned postoperatively to the preoperative value. It hovered at a depressed level in Group 2 (12.2 +/- 1.4 versus 10.2 +/- 1.1 g/dL, p < 0.001). The homologous blood requirement was significantly less in Group 1 than in Group 2 (0% versus 10%, p < 0.05). Postoperative platelet counts showed similar curves, and blood loss was not statistically significantly different between groups., Conclusion: Preoperative and intraoperative donations are safe and continue to contribute uniquely to blood conservation, providing important options in comprehensive blood conservation programs in current pediatric open-heart surgery.

A new mutant, the Wakayama epileptic rat (WER), exhibiting both spontaneous absence-like behavior and tonic-clonic convulsions, was identified in a colony of Wistar rats. To determine clear seizure characteristics of this mutant strain, we analyzed the mode of inheritance of the convulsion and observed patterns of electroencephalogram (EEG) during the seizures. F1 progeny were produced between the founder male and normal females of the same colony. Animals were monitored through the inbreeding course to analyze genetic control of epileptic behavior. EEGs were recorded using affected animals in the F3-4 and post F13 generations. After the F2 generation, affected rats spontaneously exhibited both absence-like immobile behavior and tonic-clonic convulsions. The absence-like seizures were characterized by motor arrest and head droop. The tonic-clonic convulsions began with neck and forelimb clonus, wild jumping/running, and opisthotonic posturing, and evolved to tonic, then clonic convulsions. Most convulsion onsets occurred between 25-70 days of age. Mating experiments revealed that 0%(0/18) of the animals in F1, 10%(3/26) in F2, 17%(1/6) in backcross progeny and 86% (100/116) in progeny of crosses between epileptic rats showed tonic-clonic convulsions. Ictal cortical EEGs were characterized by 4-6 (5.1 +/- 0.4, mean +/- SD) Hz spike-and-wave complexes in the absence-like seizures and by low-voltage fast waves in the tonic-clonic convulsions. This new mutant rat spontaneously exhibited both absence-like and tonic-clonic seizures. The tonic-clonic seizure was inherited as an autosomal recessive trait with 86% incidence. Thus, the new mutant rat may become a useful model for studying human inherited epilepsies.

To confirm that aspirin suppositories are an effective treatment for acute ischemic stroke, we examined the suppressive effects of 200-mg aspirin suppositories on platelet aggregation. Aspirin suppositories suppressed platelet aggregation induced by ADP or collagen, and the suppression continued for 24 h. There was no significant difference in suppression of platelet aggregation between aspirin administered by suppository and orally given aspirin. These results suggest that aspirin suppositories are a useful treatment for acute ischemic stroke., (Copyright 2003 S. Karger AG, Basel)

Bcl-2 family proteins regulate programmed cell death, and may play an important role in the selection of lymphocytes. We investigated the expression of Bcl-2, Bcl-x, Bax, Bak and Bim in human lymphocytes using flow-cytometry. Bcl-2 was down-regulated in CD4(+)8(+) (DP) thymocytes and CD19(+)38(+) tonsillar lymphocytes (GC B cells). Among DP thymocytes, cells co-expressing CD69 up-regulated Bcl-2, suggesting that the role of Bcl-2 is promoting survival of positively selected DP cells. Unexpectedly, the expression level of Bcl-x was higher in DP cells than in Single Positive (SP) cells and in CD69(+) DP thymocytes it was lower than in CD69(+) DP thymocytes. Expression of Bim was low in DP thymocytes but high in a subset of GC B cells. Bim and Bax were expressed more highly in SP than in DP thymocytes. Among peripheral blood lymphocytes (PBL), CD8(+) T cells expressed an approximately ten-fold higher level of Bcl-x than CD4(+) T cells while both subsets expressed similar levels of Bcl-2. Bak expression was low and Bim expression was absent in PBL. These results suggest that not only Bcl-2 but other members of the Bcl-2 family are involved in T cell development in the thymus and affinity maturation of B cells in the germinal center.

Estrogenic compounds such as 17beta-estradiol (E2) and its analogs are present in the aquatic environment and can adversely affect the reproductive systems of aquatic organisms. Although E2 has been detected at a maximum concentration of 64 ng/l in effluents of sewage treatment works (STWs), few reports address the associated effects on reproduction in fish. Therefore, we exposed adult medaka (Oryzias latipes) to mean measured E2 concentrations of 29.3, 55.7, 116, 227, and 463 ng/l for 21 d and assessed the effects on the egg number and fertility of paired medaka during the exposure period. In addition, we determined the hepatic vitellogenin (Vtg) concentration and histologically assessed the gonads of these fish. The number of egg produced and fertility of the paired medaka exposed to 463 ng/l E2 were significantly less compared with those of the control fish. Males in all treatment groups had developed testis-ova. Males treated with E2 concentrations = 55.7 ng/l contained relating great concentrations of hepatic Vtg. Therefore, although only the greatest E2 concentration tested in our study affected fecundity and fertility, effects of E2 were observed on induction of Vtg and testis-ova in male medaka exposed to environmentally relevant concentrations of E2.