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We evaluated the relationship between penile curvature and testosterone in Peyronie's disease patients treated in Japan.Data were obtained from 109 patients with Peyronie's disease treated with surgery at our hospital between April 2004 and December 2019. Penile deformity assessment was based on findings of a rigid erection induced by intracavernosal injection. Low total testosterone level was defined as300 ng/dl. Patients were divided into two groups according to curvature severity (I,60°; II ≥60°), then clinical factors including total testosterone were compared. Uni- and multivariate logistic regression analyses were performed to identify factors predicting severe penile deformity (≥60°).For all patients, mean total testosterone was 469 ng/dl and median curvature was 50°, with a significant inverse correlation found between curvature and testosterone level (p 0.0001). Group I and II patients numbered 55 and 54, respectively. Mean total testosterone for Group II was 397 ng/dl, significantly lower than Group I (539 ng/dl). Median curvature in 15 patients with a low testosterone level was 80°, significantly higher than those with a normal testosterone range (50°). Univariable and multivariable logistic regression analysis indicated total testosterone, follicle stimulating hormone, and C-reactive protein as significant factors correlated with severe penile deformity, among which total testosterone was most relevant.The present findings confirmed that penile deformity severity is correlated with testosterone level in Japanese males with Peyronie's disease.

Purpose To investigate the incidence, etiology, treatment indications, and outcomes regarding infertile male patients in Japan. Methods Between April, 2014 and March, 2015, the authors contacted 47 clinical specialists in male infertility who had been certified by the Japan Society for Reproductive Medicine. The participating clinicians were sent a questionnaire regarding information on their infertile patients, according to etiology and the number and success rates of male infertility operations that had been performed in their practice. Results Thirty-nine specialists returned the questionnaire and provided information regarding 7268 patients. The etiology of infertility included testicular factors, sexual disorders, and seminal tract obstruction. During the study year, the clinicians performed varicocelectomies, testicular sperm extractions (TESEs), and re-anastomoses of the seminal tract. The rate of successful varicocelectomies was >70%. The sperm retrieval rates with conventional TESE and microdissection TESE were 98.3% and 34.0%, respectively, while the patency rates with vasovasostomy and epididymovasostomy were 81.8% and 61.0%, respectively. Conclusion Surgical outcomes for infertile male patients are favorable and can be of great clinical benefit for infertile couples. To achieve this, urologists should work in collaboration with gynecological specialists in order to optimize the treatment of both partners.

We propose Timing Group Division (TGD) for faster IEEE 802.15.4 throughput. Our proposed method splits terminals into groups. All terminals in each group have both TGD delay and CSMA/CA delay. Terminals avoid packet collision by using two kinds of delay so that the system becomes faster than using only a CSMA/CA system. When terminals were divided into two groups or more, we confirmed faster communication from our actual experimental results. Our proposed method was able to communicate faster than the method using only CSMA/CA according to experimental results using 16 end-devices, with a speed increase of 40% or more. Furthermore, we propose an autonomous grouping method using RSSI for the autonomous decentralized network. Our proposed method using RSSI was faster than the method using only CSMA/CA according to actual experimental results, with a speed increase of more than 25%. Thus, we have established an autonomous grouping method for TGD based on our proposed method. © 2012 Wiley Periodicals, Inc. Electron Comm Jpn, 95(6): 41–51, 2012; Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/ecj.10429

We propose a system to estimate the traffic congestion distance using IEEE 802.15.4, that is extended by multicommunication frequency division multiplexing. Our proposed system is developed at 20% equipped rate, where five lanes are considered. Furthermore, in our proposed system, it is assumed that the equipped rate in all lanes increases. Our proposed method can estimate an error rate lower than 10% approximately at on equipped rate greater than 50%. Additionally, we use 16 terminals in the actual environment. From the actual experiment, we find that our proposed method involving the use of communication pattern 2 is 300% faster than the existing method. © 2012 Institute of Electrical Engineers of Japan. Published by John Wiley & Sons, Inc.

Stem cells have the ability both to differentiate into numerous tissues and to self‐renew. Because of these unique properties, stem cells are promising candidates for use in regenerative medicine. Among stem cell types, embryonic stem (ES) cells have been the most studied; however, alternatives such as induced pluripotent stem cells or other adult stem cells are now being established. In this review, we focus on stem cell research that may have applications in treating male infertility. Stem cells with ES‐like properties have been generated from adult human testis tissue. We expect that breakthroughs in stem cell research will increase our understanding of male infertility and lead to treatments in the near future.

Purpose Induced pluripotent stem (iPS) cells have been generated from mouse and human fibroblasts by several groups; however, transplanted mouse iPS cells can cause teratomas, depending on their tissue of origin. Therefore, human iPS cells are preferable, and various tissues are being evaluated for their potential to generate human iPS cells. Methods We examined whether adult human testicular tissue had undergone reprogramming by introducing four transcription factors, OCT4, SOX2, KLF4, and C-MYC, using lentiviral vectors. Results We obtained embryonic stem (ES)-like cells derived from human testicular tissue by introducing four cDNAs, encoding the transcription factors OCT4, SOX2, KLF4, and C-MYC, using lentiviral vectors. We showed that DAZL and VASA were expressed in testicular cells but down-regulated in ES-like cells, indicating that the cells had undergone reprogramming. ES-like cells could develop tumors, which is a hallmark of pluripotency, when SCID mice were injected with these cells. Conclusions We induced iPS cells from adult human testicular tissue by introducing four transcription factors, OCT4, SOX2, KLF4, and C-MYC, using lentiviral vectors. Future studies on these cells may elucidate the causes of male infertility, and eventually lead to treatments with autologous testicular tissue-derived iPS cells.

Scand J Caring Sci; 2011; 25; 419–425 Patient perception of nursing service quality; an applied model of Donabedian’s structure-process-outcome approach theory Background: Nursing is a labour-intensive field, and an extensive amount of latent information exists to aid in evaluating the quality of nursing service, with patients’ experiences, the primary focus of such evaluations. To effect further improvement in nursing as well as medical care, Donabedian’s structure-process-outcome approach has been applied. Aims: To classify and confirm patients’ specific experiences with regard to nursing service based on Donabedian’s structure-process-outcomes model for improving the quality of nursing care. Methods: Items were compiled from existing scales and assigned to structure, process or outcomes in Donabedian’s model through discussion among expert nurses and pilot data collection. With regard to comfort, surroundings were classified as structure (e.g. accessibility to nurses, disturbance); with regard to patient–practitioner interaction, patient participation was classified as a process (e.g. expertise and skill, patient decision-making); and with regard to changes in patients, satisfaction was classified as an outcome (e.g. information support, overall satisfaction). Patient inquiry was carried out using the finalized questionnaire at general wards in Japanese hospitals in 2005–2006. Reliability and validity were tested using psychometric methods. Results: Data from 1,810 patients (mean age: 59.7 years; mean length of stay: 23.7 days) were analysed. Internal consistency reliability was supported (α = 0.69–0.96), with factor analysis items of structure aggregated to one factor and overall satisfaction under outcome aggregated to one. The remaining items of outcome and process were distributed together in two factors. Inter-scale correlation (r = 0.442–0.807) supported the construct validity of each structure-process-outcome approach. All structure items were represented as negative-worded examples, as they dealt with basic conditions under Japanese universal health care system, and were regarded as representative related to concepts of dissatisfaction and no dissatisfaction. Conclusion: Patients’ experiences with nursing service were confirmed using Donabedian’s approach and can therefore be applied to improve quality of nursing practice by practitioners, managers and policy makers.

The secondary systems are expressed by a reduced model in trunk transmission system analysis. Thus, multiple distributed generators in the secondary system are expressed by the reduced model. But by representing distributed generators by the reduced model, partial dropout of distributed generators cannot be expressed, and therefore there is a possibility that we cannot perform an accurate analysis. Consequently, we studied a method of dropout characteristics in which distributed generators are expressed by the reduced model. To obtain the dropout characteristic, we performed parameter studies by the detailed model and the reduced model on a secondary system under the same conditions. The parameter study indicated that in the dropout characteristic the remaining voltage in the primary system is correlated with the percentage of induction machines in the secondary system. Thus, we derived a dropout characteristic in which the above two quantities were taken as parameters. To confirm the validity of this dropout characteristic, we calculated the dropout magnitudes of the distributed generator for two or more conditions with both the detailed model and the reduced model. © 2010 Wiley Periodicals, Inc. Electr Eng Jpn, 172(1): 27–37, 2010; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/eej.20869

Purpose Inappropriate intromission time causes sexual problems for couples, and therefore it is important for the couple to set treatment targets. Methods We investigated appropriate intromission times by conducting a questionnaire survey of the interval from initiation of insertion to just before ejaculation in Japanese married couples. A questionnaire survey of 300 married couples was conducted by mail. Results The estimated mean intromission times were 14.5 min (median: 10 min) for male subjects and 13.6 min (median: 10 min) for female subjects. The mean desired intromission time for female subjects was 15.7 min (median: 15 min). Regarding the difference between the actual and desired intromission times, the desired time was longer, the same, and shorter for 43.0%, 38.7%, and 18.3% of all the female subjects. Conclusions It seems that female subjects may consider a wide variety of intravaginal insertion times to be desirable. Accordingly, married couples need to improve communication regarding the desired duration of intromission and other related issues.

The expression of the neural cell adhesion molecule NCAM and its polysialic acid (PSA) moiety was documented during embryonic development and adult differentiation of chicken lens cells. In both the embryo and adult, NCAM is predominantly found in the epithelium and the zone of young elongating cells of the annular pad. NCAM abundance drops markedly in the cortical fibers and is further reduced in the lens nucleus. Epithelial cell NCAM is more highly poly-sialylated in the adult than in the embryonic lens. Three isoforms of NCAM at 180, 140, and 120 kDa were detected in the lens and predominantly associated with the unit membrane-enriched plasma membranes of fiber cells. The distribution of NCAM relative to MP26 and the adherence junction-associated glycoprotein N-cadherin suggests that NCAM could influence the formation of fiber cell gap junctions and adherence junctions.

Spermatogonial stem cells (SSCs) are self-renewing cells whose progeny are committed to differentiate into spermatozoa; this is a life-long process in male mammals. There are several methods for obtaining enriched populations of mouse SSCs, and immunological separation using surface antigens is a commonly used technique. The study of human SSCs is much less advanced. We used biopsied human testicular tissues [obstructive azoospermia patients (n = 5) and patients who underwent a testis biopsy as part of an evaluation for infertility (n = 7)] to obtain Thy-1+ cells. Thy-1—a glycosyl phosphatidylinositol-anchored surface antigen—is a marker uniquely expressed on SSCs that is used to isolate SSC-enriched cell populations in mice. The Thy-1+ cells from human testicular tissues were cultured in a basic system consisting of serum-free medium and mitotically inactivated STO (SIM mouse embryo-derived thioguanine- and ouabain-resistant) cell feeders with added growth factors: glial cell line-derived neurotrophic factor (GDNF), basic fibroblast growth factor (bFGF), and GDNF-family receptor α1 (GFRα-1). The Thy-1+ cells were maintained in vitro using this system for 1 week. The Thy-1+ cells expressed OCT3/4 and alkaline phosphatase, like mouse SSCs. They also expressed NANOG. Thy-1+ cells injected into nude mice did not cause tumor formation over a period of at least 6 months. These results support the possibility that the Thy-1+ cell population included human SSCs, and that Thy-1 may be a marker for human SSCs.

Our previous study showed that treatment of cultured bovine adrenal chromaffin cells with phorbol 12, 13-dibutyrate (PDBu) or 12-O-tetradecanoylphorbol 13-acetate (TPA) caused a rapid ( 15 h) translocation of both conventional (c) protein kinase C-α (PKC-α) and novel PKC-e (but not atypical PKC-ζ) from cytosol to membranes, whereas thymeleatoxin (TMX) increased the similar but selective membrane association of only cPKC-α. In the present study, chronic (≥12 h) treatment of chromaffin cells with PDBu raised cell surface 125I-insulin binding without altering the KD value ; it developed in a concentration (EC50 = 1.9 nM)-and time (t1/2 = 14.6 h)-dependent manner, reaching its maximum 115% increase at 48 h. Either TPA (30 nM) or TMX (EC50 = 6.4 nM) also increased 125I-insulin binding by 97 or 88%, whereas the biologically inactive 4α-TPA had no effect. The increasing effect of PDBu (30 nM for 24 h) on 125I-insulin binding was significantly blocked, even when H7, an inhibitor of PKC, was added at 8 h after the initiation of PDBu treatment. Concurrent treatment with brefeldin A, an inhibitor of vesicular transport from the trans-Golgi network, cycloheximide, an inhibitor of protein synthesis, or 5,6-dichlorobenzimidazole riboside, an inhibitor of RNA synthesis, abolished the PDBu-induced increment of 125I-insulin binding. Western blot analysis, using antibody against the β-subunit of the insulin receptor, showed that treatment with PDBu (30 nM) or TMX (EC50 = 2.3 nM) increased levels of insulin receptor precursor (~190 kDa ; t1/2 = 7.1 h) and insulin receptor β-subunit (t1/2 = 15.4 h), causing their almost maximum 52 and 59% rises, respectively, at 24 h. Northern blot analysis revealed that PDBu or TMX increased levels of insulin receptor mRNAs by ~35% as soon as 3 h, producing its monophasic peak ~76% increases at 24 h. All of these increasing effects of PDBu and TMX on 125I-insulin binding and insulin receptor β-subunit and insulin receptor mRNA levels were entirely prevented by simultaneous treatment with Go6976, a selective inhibitor of cPKC. These results suggest that long-term activation of cPKC-α up-regulates the density of the cell surface insulin receptor via transcriptional/translational events.

Aim The aim of the present study was to investigate the assessment of currently prescribed drugs and approaches to overall erectile dysfunction (ED) therapy.

In cultured bovine adrenal chromaffin cells, chronic (≥ 24 h) treatment with lysophosphatidic acid (LPA) augmented veratridine-induced 22Na+ influx via Nav1.7 by ∼22% (EC50 = 1 nmol/L), without changing nicotine-induced 22Na+ influx via nicotinic receptor-associated channel. LPA enhanced veratridine (but not nicotine)-induced 45Ca2+ influx via voltage-dependent calcium channel and catecholamine secretion. LPA shifted concentration–response curve of veratridine for 22Na+ influx upward, without altering the EC50 of veratridine. Ptychodiscus brevis toxin-3 allosterically enhanced veratridine-induced 22Na+ influx by twofold in non-treated and LPA-treated cells. Whole-cell patch-clamp analysis showed that peak Na+ current amplitude was greater by 39% in LPA (100 nmol/L for 36 h)-treated cells; however, I–V curve and steady-state inactivation/activation curves were comparable between non-treated and LPA-treated cells. LPA treatment (≥ 24 h) increased cell surface [3H]saxitoxin binding by ∼28%, without altering the Kd value; the increase was prevented by cycloheximide, actinomycin D, or Ki16425, dioctylglycerol pyrophosphate 8:0 (two inhibitors of LPA1 and LPA3 receptors), or botulinum toxin C3 (Rho inhibitor), Y27632 (Rho kinase inhibitor), consistent with LPA1 receptor expression in adrenal chromaffin cells. LPA raised Nav1.7 mRNA level by ∼37%. Thus, LPA–LPA1 receptor–Rho/Rho kinase pathway up-regulated cell surface Nav1.7 and Nav1.7 mRNA levels, enhancing veratridine-induced Ca2+ influx and catecholamine secretion.

1. The mechanisms involved in the fine adjustment of iris sphincter muscle tone are largely unknown. The aim of the present study was to clarify the effects of adrenomedullin on the resting tension of the bovine isolated iris sphincter muscle. 2. The motor activity of the bovine isolated iris sphincter muscle was measured isometrically. The effects of adrenomedullin on resting tension were analysed in the presence of indomethacin. The presence of adrenomedullin mRNA in the preparation was determined by reverse transcription-polymerase chain reaction. Immunolabelling for adrenomedullin was also performed. 3. Adrenomedullin significantly decreased the resting tension of the muscle. The relaxant effect of adrenomedullin was significantly inhibited by adrenomedullin (22-52), a putative antagonist for the adrenomedullin receptor, or calcitonin gene-related peptide (CGRP) (8-37), a putative antagonist for the CGRP1 receptor. The relaxant effect was almost completely blocked by a combination of adrenomedullin (22-52) and CGRP (8-37). 4. The relaxant effect of adrenomedullin was also significantly diminished by 2',5'-dideoxyadenosine, an inhibitor of adenylate cyclase, N(G)-nitro-L-arginine, an inhibitor of nitric oxide synthesis, or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of soluble guanylate cyclase. 5. Reverse transcription-polymerase chain reaction analysis showed that adrenomedullin mRNA was expressed in the muscle strip. Immunopositive staining for adrenomedullin was detected in blood vessel cells and in the iris sphincter muscle cells. 6. These results suggest that adrenomedullin may be an autocrine and paracrine regulator of the resting tension of the iris sphincter muscle. Its biological effects may be due to the direct involvement of adrenomedullin receptors and also to the stimulation of CGRP1 receptors. The stimulation of these receptors by the peptide leads to the activation of adenylate cyclase and soluble guanylate cyclase and subsequent relaxation of the muscle strip.

Introduction: Castleman's disease (CD), idiopathic lymph-node hyperplasia, is a heterogeneous disease of unknown origin. Although the pathophysiology is yet to be elucidated, interleukin (IL)-6 produced by swollen lymph nodes has been reported to play a crucial role in CD. Case report: This report presents a case of a 37-yr-old man with CD showing marked elevation of IL-6, hypereosinophilia, and also IL-5 elevation. To date, IL-5 has not been reported to influence CD. However, because in this case the serum concentration of IL-5 produced from swollen lymph nodes paralleled the general symptoms, IL-5, in addition to IL-6, may have played an important role in the disease. Discussion: Although in CD the serum IL-5 level is generally within the normal range, mild eosinophilia is a common complication of the disease. This case indicates that IL-5 can also influence eosinophilia. Taken together, this suggests that there may be a more general association between local production of IL-5 and the pathology of CD.

Sodium‐glucose cotransporter 2 inhibitors (SGLT2i) exert their antidiabetic effects by promoting urinary glucose excretion. Nutrition therapy is obviously important, but little is known about the interactions between SGLT2i agents and carbohydrate restriction. Therefore, we studied these interactions using an obese diabetic animal model. KK‐A y mice were pair‐fed normal chow [NC; carbohydrate: fat: protein = 65:15:20], low carbohydrate [LC; 43:42:15] or severely carbohydrate restricted diets [SR; 12:45:43] for 12 weeks. Tofogliflozin (Tofo) was administered as the SGLT2i in the NC and LC diet groups. Blood glucose levels were significantly increased in the SR group. Tofo reduced blood glucose levels significantly in the NC group during the experiment and in the LC group at 2‐6 weeks. Plasma triglycerides were markedly elevated in the SR group without Tofo, but decreased in response to Tofo administration. Hepatic triglyceride contents were not changed by the LC or the SR diet alone. However, Tofo ameliorated hepatosteatosis in NC‐fed animals. Consistent with the downregulation of stearoyl‐CoA desaturase 1, the ratio of plasma monounsaturated to saturated fatty acids was significantly reduced in the LC with Tofo and in the SR alone groups, but was not altered in the NC with Tofo group. In summary, metabolism of glucose and lipids was improved by Tofo but not by the SR diet. Furthermore, Tofo improved these parameters more effectively in the NC than in the LC diet group. These data suggest that the effects of SGLT2i are distinct from those of carbohydrate restriction and that a nonrestricted dietary carbohydrate composition is essential for SGLT2i treatment to be effective.

The density and electrical activity of cell surface voltage-dependent Na(+) channels are key determinants regulating the neuronal plasticity including development, differentiation, and regeneration. Abnormalities of Na(+) channels are associated with various neurological diseases. In this paper, we review the regulatory mechanisms of cell surface Na(+) channel expression mediated by Ca(2+) signaling pathways in cultured bovine adrenal chromaffin cells. Sustained, but not transient, elevation of intracellular Ca(2+) concentration reduced the number of cell surface Na(+) channels. The reduction of Na(+) channels was suppressed by an inhibitor of calpain, a Ca(2+)-dependent protease, and by an inhibitor of protein kinase C (PKC). The activation of conventional PKC-alpha and novel PKC-epsilon reduced cell surface Na(+) channels by the acceleration of internalization of the channels and by the increased degradation of Na(+) channel alpha-subunit mRNA, respectively. On the contrary, the activation of PKC-epsilon increased Na(+) channel beta(1)-subunit mRNA level. The inhibition of calcineurin, a Ca(2+)/calmodulin-dependent protein phosphatase 2B, by immunosuppressants upregulated cell surface Na(+) channels by both stimulating externalization and inhibiting internalization of the channels without changing Na(+) channel alpha- and beta(1)-subunit mRNA levels. Thus, the signal transduction pathways mediated by intracellular Ca(2+) modulate cell surface Na(+) channel expression via multiple Ca(2+)-dependent events, and the changes in the intracellular vesicular trafficking are the important mechanisms in the regulation of Na(+) channel expression.

Background Testicular microlithiasis is said to be characterized by calcific concretions within the seminiferous tubules. There have been reports suggesting a link between testicular microlithiasis and testicular dysfunction and tumors.

Adrenomedullin is a peptide hormone with multifunctional biological properties. Its most characteristic effects are the regulation of circulation and the control of fluid and electrolyte homeostasis through peripheral and central nervous system actions. Although adrenomedullin is a vasodilator of cerebral vasculature, and it may be implicated in the pathomechanism of cerebrovascular diseases, the source of adrenomedullin in the cerebral circulation has not been investigated thus far. We measured the secretion of adrenomedullin by radioimmunoassay and detected adrenomedullin mRNA expression by Northern blot analysis in primary cultures of rat cerebral endothelial cells (RCECs), pericytes and astrocytes. We also investigated the expression of specific adrenomedullin receptor components by reverse transcriptase-polymerase chain reaction and intracellular cAMP concentrations in RCECs and pericytes. RCECs had approximately one magnitude higher adrenomedullin production (135 +/- 13 fmol/10(5) cells per 12 h; mean +/- SD, n = 10) compared to that previously reported for other cell types. RCECs secreted adrenomedullin mostly at their luminal cell membrane. Adrenomedullin production was not increased by thrombin, lipopolysaccharide or cytokines, which are known inducers of adrenomedullin release in peripheral endothelial cells, although it was stimulated by astrocyte-derived factors. Pericytes had moderate, while astrocytes had very low basal adrenomedullin secretion. In vivo experiments showed that adrenomedullin plasma concentration in the jugular vein of rats was approximately 50% higher than that in the carotid artery or in the vena cava. Both RCECs and pericytes, which are potential targets of adrenomedullin in cerebral microcirculation, expressed adrenomedullin receptor components, and exhibited a dose-dependent increase in intracellular cAMP concentrations after exogenous adrenomedullin administration. Antisense oligonucleotide treatment significantly reduced adrenomedullin production by RCECs and tended to decrease intraendothelial cAMP concentrations. These findings may suggest an important autocrine and paracrine role for adrenomedullin in the regulation of cerebral circulation and blood-brain barrier functions. Cerebral endothelial cells are a potential source of adrenomedullin in the central nervous system, where adrenomedullin can also be involved in the regulation of neuroendocrine functions.

1. Long-term (> or = 12 h) treatment of cultured bovine adrenal chromaffin cells with A23187 (a Ca(2+) ionophore) or thapsigargin (TG) [an inhibitor of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)] caused a time- and concentration-dependent reduction of cell surface [(3)H]-saxitoxin (STX) binding capacity, but did not change the K:(D:) value. In A23187- or TG-treated cells, veratridine-induced (22)Na(+) influx was reduced (with no change in veratridine EC(50) value) while it was enhanced by alpha-scorpion venom, beta-scorpion venom, or Ptychodiscus brevis toxin-3, like in nontreated cells. 2. The A23187- or TG-induced decrease of [(3)H]-STX binding was diminished by BAPTA-AM. EGTA also inhibited the decreasing effect of A23187. A23187 caused a rapid, monophasic and persistent increase in intracellular concentration of Ca(2+) ([Ca(2+)](i)) to a greater extent than that observed with TG. 2,5-Di-(t-butyl)-1,4-benzohydroquinone (DBHQ) (an inhibitor of SERCA) produced only a rapid monophasic increase in [Ca(2+)](i), without any effect on [(3)H]-STX binding. 3. Reduction in [(3)H]-STX binding capacity induced by A23187 or TG was attenuated by Go6976 (an inhibitor of conventional protein kinase C) or calpastatin peptide (an inhibitor of calpain). When the internalization rate of cell surface Na(+) channels was measured in the presence of brefeldin A (an inhibitor of vesicular exit from the trans-Golgi network), A23187 or TG accelerated the reduction of [(3)H]-STX binding capacity. 4. Six hours treatment with A23187 lowered Na(+) channel alpha- and beta(1)-subunit mRNA levels, whereas TG had no effect. 5. These results suggest that elevation of [Ca(2+)](i) caused by A23187, TG or DBHQ exerted differential effects on down-regulation of cell surface functional Na(+) channels and Na(+) channel subunit mRNA levels.

In cultured bovine adrenal chromaffin cells, NS-7 [4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy) pyrimidine hydrochloride], a newly-synthesized neuroprotective drug, inhibited veratridine-induced 22Na+ influx via voltage-dependent Na+ channels (IC50=11.4 μM). The inhibition by NS-7 occurred in the presence of ouabain, an inhibitor of Na+,K+ ATPase, but disappeared at higher concentration of veratridine, and upon the washout of NS-7. NS-7 attenuated veratridine-induced 45Ca2+ influx via voltage-dependent Ca2+ channels (IC50=20.0 μM) and catecholamine secretion (IC50=25.8 μM). Chronic (⩾12 h) treatment of cells with NS-7 increased cell surface [3H]-STX binding by 86% (EC50=10.5 μM; t1/2=27 h), but did not alter the KD value; it was prevented by cycloheximide, an inhibitor of protein synthesis, or brefeldin A, an inhibitor of vesicular transport from the trans-Golgi network, but was not associated with increased levels of Na+ channel α- and β1-subunit mRNAs. In cells subjected to chronic NS-7 treatment, 22Na+ influx caused by veratridine (site 2 toxin), α-scorpion venom (site 3 toxin) or β-scorpion venom (site 4 toxin) was suppressed even after the extensive washout of NS-7, and veratridine-induced 22Na+ influx remained depressed even at higher concentration of veratridine; however, either α- or β-scorpion venom, or Ptychodiscus brevis toxin-3 (site 5 toxin) enhanced veratridine-induced 22Na+ influx as in nontreated cells. These results suggest that in the acute treatment, NS-7 binds to the site 2 and reversibly inhibits Na+ channels, thereby reducing Ca2+ channel gating and catecholamine secretion. Chronic treatment with NS-7 up-regulates cell surface Na+ channels via translational and externalization events, but persistently inhibits Na+ channel gating without impairing the cooperative interaction between the functional domains of Na+ channels. Keywords: Neuroprotective drugs, sodium channels, up-regulation, [3H]-saxitoxin binding, Northern blot, veratridine, 22Na+ influx, 45Ca2+ influx, catecholamine secretion, adrenal chromaffin cells Introduction It has become increasingly evident that noninactivating Na+ currents (Taylor, 1993) via voltage-dependent Na+ channels (Catterall, 1992) may initiate the detrimental cascade of hypoxia/ischaemia-induced cell injury, such as the massive overflow of glutamate and catecholamines (Toner & Stamford, 1997), the intracellular Ca2+ overload via reversed operation of Na+-Ca2+ exchanger (Obrenovitch & Richards, 1995; Urenjak & Obrenovitch, 1996), as well as cytotoxic formation of nitric oxide (NO) (Strijbos et al., 1996). A few studies have revealed that during hypoxia, the steady-state inactivation of Na+ currents was shifted to a more hyperpolarizing potential in human cortical neurons (Cummins et al., 1993) and rat hippocampus (O'Reilly et al., 1997), presumably as the compensatory defensive response against hypoxia-induced neuronal injury. Also, density of cell surface Na+ channels was fluctuated in brain during hypoxia (Perez-Pinzon et al., 1992; Xia & Haddad, 1994; 1999). NS-7 [4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy) pyrimidine hydrochloride] is a newly-synthesized neuroprotective agent. Previous in vivo and in vitro studies in cerebral cortex have shown that NS-7 attenuated hypoxia-induced degradation of cytoskeletal protein fodrin (Takagaki et al., 1997) and cell injury (Tatsumi et al., 1998b) at 10–30 μM, and the cellular mechanisms of NS-7 for neuroprotection are postulated to be attributed to the blockade of voltage-dependent Na+ and Ca2+ channels. In brain, NS-7 bound with a Ki value of 1 μM (Shimidzu et al., 1997) to the toxin binding site 2 of the Na+ channel α-subunit, a major subunit forming the ion-pore and the toxin binding sites 1–5 (Catterall, 1992); NS-7 at 10–30 μM diminished the overflow of glutamate (Shimidzu et al., 1997) and dopamine (Itoh et al., 1998) caused by veratridine, a toxin that interacts with the site 2 in the transmembrane segment 6 of domain I (IS6) of the Na+ channel α-subunit (Trainer et al., 1996) and activates Na+ channels (Catterall, 1992). NS-7 suppressed depolarization-elicited Na+ currents with an IC50 of 7.8 μM in NG108-15 cells (Suma et al., 1997). Also, in NG108-15 cells, NS-7 decreased Ca2+ currents via L-, N-, and T-type Ca2+ channels with IC50 values of 7.3, 4.5, and 17.1 μM, respectively (Suma et al., 1997). In cerebrocortical neurons, NS-7 attenuated high K+- or veratridine-induced activation of NO synthase at 10–30 μM presumably by inhibiting Ca2+ influx via L- and P/Q-type Ca2+ channels (Tatsumi et al., 1998a; Oka et al., 1999). NS-7 inhibited high K+-induced dopamine secretion at 10 μM (Itoh et al., 1998), but did not affect high K+-induced glutamate release in brain (Shimidzu et al., 1997). Na+ channel α-subunits arise from multiple genes and their alternative splicing (Dietrich et al., 1998), whereas the β1-subunits are structurally similar among various tissues (Makita et al., 1994). In adrenal chromaffin cells (embryologically derived from the neural crest), Na+ channel α-subunit (Yamamoto et al., 1997) is homologous to the tetrodotoxin (TTX)/saxitoxin (STX) (site 1 toxin)-sensitive human neuroendocrine type Na+ channel α-subunit (hNE-Na) (Klugbauer et al., 1995). Previous studies showed that veratridine-induced Na+ influx via Na+ channels, and the subsequent depolarization increases Ca2+ influx via voltage-dependent Ca2+ channels (Wada et al., 1985a,1985b; Lopez et al., 1995), thereby triggering veratridine-induced exocytic secretion of catecholamines (Ito et al., 1980). We found that chronic treatment of chromaffin cells with the antiepileptic drug valproic acid raised Na+ channel α- and β1-subunit mRNA levels, as well as cell surface [3H]-STX binding, thus enhancing veratridine-induced 22Na+ influx, 45Ca2+ influx and catecholamine secretion (Yamamoto et al., 1997). Similar observations were made in long-term treatment of chromaffin cells with the anticonvulsant carbamazepine (Yoshimura et al., 1998). Also, chronic in vivo and in vitro treatment with the antiepileptic drug phenytoin (Sashihara et al., 1994) and the antiarrhythmic drug mexiletine (Kang et al., 1997) caused up-regulation of Na+ channels, thereby modifying neuronal and cardiac pathophysiologies. Our present study examined whether/how short- and long-term treatment of chromaffin cells with NS-7 might alter 22Na+ influx, 45Ca2+ influx and catecholamine secretion, using veratridine, α-scorpion venom (site 3 toxin), β-scorpion venom (site 4 toxin) and Ptychodiscus brevis toxin-3 (PbTx-3) (site 5 toxin) (Wada et al., 1987; 1992; Catterall, 1992). Also, the effects of chronic treatment with NS-7 on cell surface [3H]-STX binding, Na+ channel α- and β1-subunit mRNA levels were evaluated.

Exocytotic responses associated with phagocytosis were investigated in a single neutrophil with a special reference to their dynamic properties and their spatiotemporal relationships with ionic and chemical responses during phagocytosis. The real-time sequence of phagocytosis-exocytosis was directly visualized by video-enhanced contrast differential interference contrast (VEC-DIC) microscopy. The actual release of contents from such a granule was proven by examining a cell loaded with quinacrine with a dual imaging system that allowed us to observe DIC and fluorescence images simultaneously at a high magnification. During the process of phagosome formation in a neutrophil engulfing an opsonized zymosan, the exocytotic response was observed first in a granule located near the cell surface initially attached to the zymosan, and then in other granules sequentially along pseudopodia surrounding the zymosan. When the phagocytosis was induced in a medium containing luminol, a chemiluminescence due to active oxidants was detected exclusively in the region of phagosome, suggesting that exocytosis took place on the phagosomal membrane and not on the plasma membrane. Changes in cytosolic free calcium concentration ([Ca2+]i) were further measured using fura-2 under the dual imaging system. [Ca2+]i transients were more closely related to the extension of pseudopodia for engulfing zymosan and not directly to the exocytosis. These findings lead to a conclusion that exocytosis associated with phagocytosis is initiated by attachment of the cell membrane to the invading organism and mediated by local activation of the phagosomal membrane.

This paper investigates the phase retrieval holography (PRH) method using the Misell algorithm for measuring the surface error of a large reflector antenna. PRH is a technique that does not require phase measurement. The algorithm is investigated experimentally and the estimation errors are evaluated quantitatively using measurement simulation. First, it is shown that estimation errors can be reduced using an initial aperture distribution calculated by ray-tracing. The measurement resolution is improved by applying an extrapolation algorithm to the Misell algorithm. Finally, the surface error of a Cassegrain antenna with an aperture of 64 m to resolution of about 4.1 m in the aperture plane was measured by receiving a test signal from a geostationary satellite (frequency: 1.545 GHz). As a result of the experiment, the surface error of 1.57 mm RMS and measurement reproducibility of 0.54 mm RMS were obtained and it was confirmed that this method is effective as a simple surface measurement method.

To examine hemodynamic, plasma catecholamines, and cyclic AMP changes during tilt in patients with neurocardiogenic (vasodepressor) syncope, six patients underwent 80 degrees head-up tilt test for 10 minutes with isoproterenol infusion (1-3 micrograms/min). Venous blood was sampled in the supine position, at 3 minutes of tilt, and at the onset of vasodepressor reaction during tilt. All patients had previous tilt studies in which vasodepressor syncope had been induced reproducibly (mean 3.3 episodes in each patient). Syncope was induced at 6.1 +/- 0.4 minutes of tilt with an infusion of isoproterenol (mean 1.7 +/- 0.3 micrograms/min). Although arterial pressure and heart rate did not change significantly between in the supine position and at 3 minutes of tilt, plasma norepinephrine increased significantly at 3 minutes of tilt (0.44 +/- 0.10 ng/mL; P < 0.05) and at the onset of vasodepressor reaction (0.49 +/- 0.12 ng/mL; P < 0.01) compared to the supine position with isoproterenol (0.34 +/- 0.10 ng/mL). Also, cyclic AMP (cAMP) increased significantly at 3 minutes of tilt (25.3 +/- 2.0 pmol/mL; P < 0.005) and at the onset of vasodepressor reaction (29.6 +/- 1.7 pmol/mL; P < 0.005) compared to the supine position with isoproterenol (20.4 +/- 1.9 pmol/mL). After administration of selective beta 1-blocker, metoprolol (40 mg/day), plasma norepinephrine, and cAMP during tilt did not change significantly compared to baseline tilt. However, metoprolol prevented the syncope in 3 of 6 patients. After administration of beta 1-, beta 2-blocker, propranolol (30 mg/day), cAMP at 3 minutes of tilt decreased significantly compared to the baseline tilt (16.9 +/- 1.4 pmol/mL vs 25.3 +/- 2.0 pmol/mL; P < 0.05) and propranolol prevented the syncope in all six patients. We concluded that the increase of cAMP may play an important role for the induction of vasodepressor reaction in patients with neurocardiogenic (vasodepressor) syncope. The concentration of cAMP showed more sensitive response to vasodepressor reaction than that of norepinephrine.

In cultured bovine adrenal medullary cells, stimulation of nicotinic receptors by carbachol evoked the Ca 2+ -dependent exocytotic cosecretion of proadrenomedullin N-terminal 20 peptide (PAMP) (EC 50 =50.1 μM) and catecholamines (EC 50 =63.0 μM), with the molar ratio of PAMP/catecholamines secreted being equal to the ratio in the cells. Addition of PAMP[1-20]NH 2 inhibited carbachol-induced 22 Na + influx via nicotinic receptors (IC 50 =2.5 μM) in a noncompetitive manner and thereby reduced carbachol-induced 45 Ca 2+ influx via voltage-dependent Ca 2+ channels (IC 50 =1.0 μM) and catecholamine secretion (IC 50 =1.6 μM). It did not alter high K + -induced 45 Ca 2+ influx via voltage-dependent Ca 2+ channels or veratridine-induced 22 Na + influx via voltage-dependent Na + channels. PAMP seems to be a novel antinicotinic peptide cosecreted with catecholamines by a Ca 2+ -dependent exocytosis in response to nicotinic receptor stimulation

In the present study, we investigated whether activation of the MAPK family could regulate the cell surface expression of Na channels in cultured bovine adrenal chromaffin cells. The results suggest that constitutively activated ERK (but not p38 or JNK), by various extracellular stimuli, down-modulates the density of cell surface Na channels, which was mediated via the destabilization of Na channel alpha-subunit mRNA.

In a radio interferometer, the error of the antenna position is one of the factors affecting significantly the synthesized map. We have developed a calibration method for the antenna position for the Nobeyama millimeter array. In comparison with the conventional method, the new calibration method has two significant characteristics. First, using the relative phase between reference celestial radio sources, the phase variation of a large time scale due to the atmospheric fluctuation and the receiver variation can be eliminated. Second, by means of the three-dimensional discrete Fourier transform, one can estimate the antenna position errors without any ambiguity. With this calibration method, the antenna positions of the Nobeyama millimeter array were determined within 0.3 mm rms. As a result, directions of celestial radio sources were measured within 0.1 arcsec.

A novel core–shell composite photocatalyst, commercially available titanium(IV) oxide (TiO2) particles directly incorporated into a hollow amorphous silica shell, was fabricated by successive coating of TiO2 with a carbon layer and a silica layer followed by heat treatment to remove the carbon layer. The composite induced efficient photocatalytic reactions when relatively small substrates were used, such as methanol dehydration and decomposition of acetic acid, without any reduction in the intrinsic activity of original TiO2, but did not exhibit efficient photocatalytic activity for decomposition of large substrates, methylene blue and polyvinyl alcohol. The unique size-selective properties of the composites are due to their structural characteristics, i.e., the presence of a pore system and a void space in the silica shell and between the shell and medial TiO2 particles, respectively. The loading of alkylsilyl groups on the surface of the composite led to highly photostable floatability: the floated sample also induced efficient photocatalytic reaction for decomposition of acetic acid while retaining floatation at the gas/water interface.

We investigated the presence of Na+/H+ exchange in cultured bovine adrenal medullary cells. The intracellular pH in control cells measured by 5,5-dimethyl[2-14C]oxazolidine-2,4-dione was 7.13 +/- 0.02 (n = 6). Removal of Na+ from the incubation medium shifted the intracellular pH down to 6.67 +/- 0.12 (n = 6). Reintroduction of Na+ to the medium caused a rapid recovery in intracellular pH to 7.20-7.30 that was associated with an increase in uptake of 22Na+ by the cells. Both increases in intracellular pH and uptake of 22Na+ were inhibited by amiloride, an inhibitor of Na+/H+ exchange. The recovery of intracellular pH by addition of Na+ was partially inhibited by quinidine, another inhibitor of Na+/H+ exchange, but not by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, an anion-exchange (Cl-/HCO3-) inhibitor. Li+ could substitute for Na+ in the recovery of intracellular pH. Carbachol caused an increase in intracellular pH from 7.12 +/- 0.01 to 7.21 +/- 0.02 (n = 10). This increase in intracellular pH caused by carbachol was inhibited by amiloride. These results suggest the existence of an amiloride-sensitive Na+/H+ exchange that regulates the intracellular pH in adrenal medullary cells.

Our previous study showed that treatment of cultured bovine adrenal chromaffin cells with phorbol 12, 13-dibutyrate (PDBu) or 12-O-tetradecanoylphorbol 13-acetate (TPA) caused a rapid ( 15 h) translocation of both conventional (c) protein kinase C-alpha (PKC-alpha) and novel PKC-epsilon (but not atypical PKC-zeta) from cytosol to membranes, whereas thymeleatoxin (TMX) increased the similar but selective membrane association of only cPKC-alpha. In the present study, chronic (>/=12 h) treatment of chromaffin cells with PDBu raised cell surface (125)I-insulin binding without altering the K(D) value; it developed in a concentration (EC(50) = 1.9 nM)-and time (t(1/2) = 14.6 h)-dependent manner, reaching its maximum 115% increase at 48 h. Either TPA (30 nM) or TMX (EC(50) = 6.4 nM) also increased (125)I-insulin binding by 97 or 88%, whereas the biologically inactive 4alpha-TPA had no effect. The increasing effect of PDBu (30 nM for 24 h) on (125)I-insulin binding was significantly blocked, even when H7, an inhibitor of PKC, was added at 8 h after the initiation of PDBu treatment. Concurrent treatment with brefeldin A, an inhibitor of vesicular transport from the trans-Golgi network, cycloheximide, an inhibitor of protein synthesis, or 5,6-dichlorobenzimidazole riboside, an inhibitor of RNA synthesis, abolished the PDBu-induced increment of (125)I-insulin binding. Western blot analysis, using antibody against the beta-subunit of the insulin receptor, showed that treatment with PDBu (30 nM) or TMX (EC(50) = 2.3 nM) increased levels of insulin receptor precursor (approximately 190 kDa; t(1/2) = 7.1 h) and insulin receptor beta-subunit (t(1/2) = 15.4 h), causing their almost maximum 52 and 59% rises, respectively, at 24 h. Northern blot analysis revealed that PDBu or TMX increased levels of insulin receptor mRNAs by approximately 35% as soon as 3 h, producing its monophasic peak approximately 76% increases at 24 h. All of these increasing effects of PDBu and TMX on (125)I-insulin binding and insulin receptor beta-subunit and insulin receptor mRNA levels were entirely prevented by simultaneous treatment with Go6976, a selective inhibitor of cPKC. These results suggest that long-term activation of cPKC-alpha up-regulates the density of the cell surface insulin receptor via transcriptional/translational events.

Angiotensin-converting enzyme (ACE) activity was measured in microvessels prepared from cerebral cortices of 4-week-old spontaneously hypertensive rats (SHR). The Vmax value of the ACE activity in the cerebral microvessels of SHR was lower than that of Wistar Kyoto controls of the same age by 25% without difference in Km value for substrate. The low activity of ACE in the cerebral microvessels of young SHR indicates that in this animal model of hypertension the function of ACE is genetically altered in the cerebral microvessels, which may be correlated with the alteration of the cerebral microcirculation and pathogenesis of hypertension.