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Introduction Sexual problems have been more prevalent among East Asian women than those from other areas of the world. However, Japanese women seldom tend to consult their treating physicians as such intimate problems are socially awkward topics to share and may be considered shameful. Presently, there is little data in the literature regarding women's sexual problems in Japan. Aims We aimed (i) to investigate the types of sexual problems that were reported among Japanese women who had sought online consultations; and (ii) to examine whether factors such as age and family structure (marital status and presence of children) increased the likelihood of sexual problems. Methods An online helpline received a total of 316 messages from Japanese women related to sexual problems over a 3-year period. We evaluated 276 respondents, who provided demographic information such as age and family structure as well as their response to an open-ended question regarding their sexual problems. Main Outcome Measures Main outcome measures were the types of sexual problems reported by Japanese women. Results The majority of respondents were in their 30s (53.6%). Sexual aversion accounted for 42.4% of the complaints, partners' sexual issues for 18.5%, and pain during sex for 16.7%. Family structure significantly correlated with sexual problems (P Conclusion Sexual aversion was the most common sexual problem among Japanese women who sought help via the online helpline. Family structure was related to sexual problems. More detailed assessments of family structure may be important in better identifying the triggering causes of the reported sexual problems.

Teratomas rarely present as a pediatric congenital primary penile mass. We describe a 14-month-old boy with a blister-like mass on his distal left penis. The subcutaneous mass measured 1.5 cm (length) × 1.0 cm (width) × 1.2 cm (height) on ultrasonography. There were clear margins between these structures and the lesion. At the age of 5 years, he received an extirpation surgery. Histologic analysis revealed that it was a mature teratoma. In our view, surgical resection should be the treatment of choice for a pediatric penile mass with the alertness of teratomas because of the possibility of malignant alteration and invasion of adjacent structures till unresectable.

TiO2 particles encapsulated in hollow silica shells with controlled shell thicknesses were fabricated. Compared to naked TiO2 without coverage by a hollow silica shell, all of these composites showed low levels of photocatalytic activity for gas-phase decomposition of acetone into CO2. The activities over these composites tended to decrease with increase in thickness of the lateral silica shell. These results can be explained by a decrease in the diffusion of acetone with increase in thickness of the silica shell, leading to hampering of supply of the substrate to the surfaces of core TiO2 particles. In contrast, for the overall photodecomposition of gaseous 2-propanol into CO2, the presence of the lateral silica shell was found to be beneficial, i.e., the composite induced a high level of activity compared to the activity of naked TiO2. The enhancement of activity for this reaction was likely to be due to condensation of the intermediate product of acetone in the void spaces, leading to an increase in the collision probability between acetone and the surface of the TiO2 core.

In cultured bovine adrenal chromaffin cells expressing Na(V)1.7 isoform of voltage-dependent Na(+) channels, we have previously reported that lithium chloride (LiCl) inhibits function of Na(+) channels independent of glycogen synthase kinase-3 (GSK-3) (Yanagita et al., 2007). Here, we further examined the effects of chronic lithium treatment on Na(+) channels. LiCl treatment (1-30 mM,or = 12 h) increased cell surface [(3)H]saxitoxin ([(3)H]STX) binding by approximately 32% without altering the affinity of [(3)H]STX binding. This increase was prevented by cycloheximide and actinomycin D. SB216763 and SB415286 (GSK-3 inhibitors) also increased cell surface [(3)H]STX binding by approximately 31%. Simultaneous treatment with LiCl and SB216763 or SB415286 did not produce an increased effect on [(3)H]STX binding compared with either treatment alone. LiCl increased Na(+) channel alpha-subunit mRNA level by 32% at 24 h. LiCl accelerated alpha-subunit gene transcription by 35% without altering alpha-subunit mRNA stability. In LiCl-treated cells, LiCl inhibited veratridine-induced (22)Na(+) influx as in untreated cells. However, washout of LiCl after chronic treatment enhanced veratridine-induced (22)Na(+) influx, (45)Ca(2+) influx and catecholamine secretion by approximately 30%. Washout of LiCl after 24 h treatment shifted concentration-response curve of veratridine upon (22)Na(+) influx upward, without altering its EC(50) value. Ptychodiscus brevis toxin-3 allosterically enhanced veratridine-induced (22)Na(+) influx by two-fold in untreated and LiCl-treated cells. Whole-cell patch-clamp analysis indicated that I-V curve and steady-state inactivation/activation curves were comparable between untreated and LiCl-treated cells. Thus, GSK-3 inhibition by LiCl up-regulated cell surface Na(V)1.7 via acceleration of alpha-subunit gene transcription, enhancing veratridine-induced Na(+) influx, Ca(2+) influx and catecholamine secretion.

A composite of titanium(IV) oxide (TiO 2 ) particles (core) and nanoporous silica (shell) was prepared by successive coating of a carbon layer and an octadecyl-functionalized silica layer on TiO 2 , followed by heat treatment to remove the organic components. Transmission electron microscope (TEM) observation and nitrogen (N 2 ) sorption analyses showed that the composite has a unique rattle-type structure, i.e., TiO 2 particles were encapsulated in the hollow silica shell having well-developed porosity. When the photocatalytic activity for gas phase decomposition of acetone over the composite was compared with that over naked TiO 2 without the lateral silica shell, the activity over the composite tended to become higher than that over naked TiO 2 as the initial amount of acetone in the system was reduced. The enhancement of decomposition rate under a diluted condition was due to condensation of acetone on the lateral silica shell, which resulted in enhancement of the collision rate between the substrate and the surface of the TiO 2 core.

The ability of calcineurin to regulate IRS-1 and IRS-2 levels has not been examined in any given cells, although calcineurin inhibition by therapeutic immunosuppressants produced cytoprotective and cytotoxic effects (e.g., new-onset of diabetes mellitus, seizure). Chronic (>or=3h) treatment of cultured bovine adrenal chromaffin cells with cyclosporin A or FK506 decreased IRS-2 protein level by approximately 50% (IC(50)=200 or 10nM), without changing IRS-2 mRNA level, and insulin receptor, insulin-like growth factor-I (IGF-I) receptor, IRS-1, PI3K/PDK-1/Akt/GSK-3beta and ERK1/ERK2 protein levels. When the cells were washed to remove the test drug, the decreased IRS-2 level restored to the control level. Cyclosporin A or FK506 treatment inhibited calcineurin activity (IC(50)=500 or 40 nM, in vitro assay). Rapamycin, an FK506-binding protein ligand unable to inhibit calcineurin, failed to decrease IRS-2, but reversed FK506-induced decreases of calcineurin activity and IRS-2 level. Pulse-label followed by polyacrylamide gel electrophoresis revealed that cyclosporin A or FK506 accelerated IRS-2 degradation rate (t(1/2)) from >24 to approximately 4.2h, without altering IRS-2 synthesis. IRS-2 reduction by cyclosporin A or FK506 was prevented by lactacystin (proteasome inhibitor), but not by calpeptin (calpain inhibitor) or leupeptin (lysosome inhibitor). Cyclosporin A or FK506 increased serine-phosphorylation and ubiquitination of IRS-2. Cell surface (125)I-IGF-I binding capacity was not changed in cyclosporin A- or FK506-treated cells; however, IGF-I-induced phosphorylations of GSK-3beta and ERK1/ERK2 were attenuated by approximately 50%, which were prevented by rapamycin or lactacystin. Thus, calcineurin inhibition decreased IRS-2 level via proteasomal IRS-2 degradation, attenuating IGF-I-induced GSK-3beta and ERK pathways.

Background Acyl-coenzyme A:cholesterol O -acyltransferase-1 (ACAT-1), a major ACAT isozyme in macrophages, plays an essential role in foam cell formation in atherosclerotic lesions. However, whether pharmacological inhibition of macrophage ACAT-1 causes exacerbation or suppression of atherosclerosis is controversial. Methods and results We developed and characterized a novel ACAT inhibitor, K-604. The IC 50 values of K-604 for human ACAT-1 and ACAT-2 were 0.45 and 102.85μmol/L, respectively, indicating that K-604 is 229-fold more selective for ACAT-1. Kinetic analysis indicated that the inhibition was competitive with respect to oleoyl-coenzyme A with a K i value of 0.378μmol/L. Exposure of human monocyte-derived macrophages to K-604 inhibited cholesterol esterification with IC 50 of 68.0nmol/L. Furthermore, cholesterol efflux from THP-1 macrophages to HDL 3 or apolipoprotein A-I was enhanced by K-604. Interestingly, administration of K-604 to F1B hamsters on a high-fat diet at a dose of ≥1mg/kg suppressed fatty streak lesions without affecting plasma cholesterol levels. Conclusions K-604, a potent and selective inhibitor of ACAT-1, suppressed the development of atherosclerosis in an animal model without affecting plasma cholesterol levels, providing direct evidence that pharmacological inhibition of ACAT-1 in the arterial walls leads to suppression of atherosclerosis.

Water homeostasis in the brain is essential for brain function. We have studied how aquaporin (AQP) 1 expression in GP8 immortalized rat brain microvascular endothelial cells is regulated by glucocorticoid. AQP1 protein level was raised by dexamethasone treatment in a time- and concentration-dependent manner. The up-regulation of AQP1 protein by dexamethasone was associated with an increase of AQP1 mRNA level, with no change in the degradation rate of AQP1 mRNA. AQP1 was concentrated in detergent-insoluble fractions in the cells treated with or without dexamethasone, suggesting that function/trafficking of AQP1 may be regulated via the interaction with lipid rafts. Since glucocorticoid therapy has well known beneficial effects in the treatment of brain edema, the induction of AQP1 by dexamethasone raises a possibility that AQP1 plays a role in ameliorating brain edema.

The degradation and sorting of cytoplasmic and cell-surface proteins are crucial steps in the control of cellular functions. We previously identified three mammalian Vps (vacuolar protein sorting) proteins, Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) and signal transducing adaptor molecule (STAM) 1 and -2, which are tyrosine-phosphorylated upon cytokine/growth factor stimulation. Hrs and the STAMs each contain a ubiquitin-interacting motif and through formation of a complex are involved in the vesicle transport of early endosomes. To explore the mechanism and cellular function of this complex in mammalian cells, we established an Hrs-defective fibroblastoid cell line (hrs(-/-)); embryos with this genotype died in utero. In the hrs(-/-) cells only trace amounts of STAM1 and STAM2 were detected. Introduction of wild-type Hrs or an Hrs mutant with an intact STAM binding domain (Hrs-dFYVE) fully restored STAM1 and STAM2 expression, whereas mutants with no STAM binding ability (Hrs-dC2, Hrs-dM) failed to express the STAMs. This regulated control of STAM expression by Hrs was independent of transcription. Interestingly, STAM1 degradation was mediated by proteasomes and was partially dependent on the ubiquitin-interacting motif of STAM1. Revertant Hrs expression in hrs(-/-) cells not only led to the accumulation of ubiquitinated proteins, including intracytoplasmic vesicles, but also restored STAM1 levels in early endosomes and eliminated the enlarged endosome phenotype caused by the absence of Hrs. These results suggest that Hrs is a master molecule that controls in part the degradation of STAM1 and the accumulation of ubiquitinated proteins.

We examined the role of osteopontin (OPN) in the osteoclastogenesis of arthritis using collagen-induced arthritis (CIA). Cells from arthritic joints of wild-type (OPN +/+) mice spontaneously developed bone-resorbing osteoclast-like cells (OCLs). The cultured cells showed an enhanced expression of receptor activator of nuclear factor kappaB ligand (RANKL) and a decreased expression of osteoprotegerin (OPG). The addition of OPG reduced the number of OCLs, indicating that the osteoclastogenesis depends on the RANK/RANKL/OPG system. The cells also produced OPN abundantly and anti-OPN neutralizing antibodies suppressed the development of OCLs. Moreover, the addition of OPN increased the expression of RANKL and augmented differentiation of OCLs from OPN-deficient (OPN -/-) cells. OPN, like the combination of 1alpha,25-dihydroxyvitamin D(3) and dexamethasone, also enhanced the RANKL expression and decreased OPG expression in a stromal cell line, ST2. These results suggest that OPN acts as a positive regulator in the osteoclastogenesis of arthritis through the RANK/RANKL/OPG system.

The family 10 xylanase from Streptomyces olivaceoviridis E-86 (SoXyn10A) consists of a GH10 catalytic domain, which is joined by a Gly/Pro-rich linker to a family 13 carbohydrate-binding module (CBM13) that interacts with xylan. To understand how GH10 xylanases and CBM13 recognize decorated xylans, the crystal structure of SoXyn10A was determined in complex with α-l-arabinofuranosyl- and 4-O-methyl-α-d-glucuronosyl-xylooligosaccharides. The bound sugars were observed in the subsites of the catalytic cleft and also in subdomains α and γ of CBM13. The data reveal that the binding mode of the oligosaccharides in the active site of the catalytic domain is entirely consistent with the substrate specificity and, in conjunction with the accompanying paper (Pell, G., Taylor, E. J., Gloster, T. M., Turkenburg, J. P., Fontes, C. M. G. A., Ferreira, L. M. A., Nagy, T., Clark, S. J., Davies, G. J., and Gilbert, H. J. (2004) J. Biol. Chem. 279, 9597-9605), demonstrate that the accommodation of the side chains in decorated xylans is conserved in GH10 xylanases of SoXyn10A against arabinoglucuronoxylan. CBM13 was shown to bind xylose or xylooligosaccharides reversibly by using nonsymmetric sugars as the ligands. The independent multiple sites in CBM13 may increase the probability of substrate binding.

Adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP) are multi-functional peptides derived from the same precursor, proadrenomedullin. We have studied the regulatory mechanism of expression of these peptides during neuronal differentiation of rat pheochromocytoma PC12 cells by nerve growth factor (NGF). The cellular levels of the peptides increased slightly, and then progressively decreased below the control by NGF. Immunoreactive (ir)-AM in the medium was transiently increased by NGF. Cytochemical staining showed that ir-AM and ir-PAMP were abundantly present in cytoplasm in the undifferentiated cells, and were decreased during culture with NGF. There was no preferential localization of ir-AM or ir-PAMP in neurites in comparison with in cytoplasm in the differentiated cells. Northern blot analysis showed that mRNA encoding these peptides, as detected as a band of 1.6 kb, increased more than three-fold at 1 h after the addition of NGF and then progressively decreased to one fifth of the control during 72 h. Degradation rate of the mRNA was slowed by NGF even when mRNA level is decreased after 72 h of NGF treatment. The transcription rate of their gene increased transiently and then decreased by the long-term treatment with NGF. These results demonstrate that expression of AM and PAMP is regulated by NGF along with time-dependent differentiation: AM gene transcription is transiently activated by NGF, whereas it was suppressed during neuronal differentiation of the cells.

In cultured bovine adrenal chromaffin cells, (+/-)-bupivacaine inhibited veratridine-induced 22Na(+) influx (IC(50) 6.8 microM). The IC(50) of (+)-bupivacaine (2.8 microM) was 6.2-, 7.4-, and 17.1-fold lower than those of (-)-bupivacaine (17.3 microM), (-)-ropivacaine (20.6 microM), and lidocaine (47.8 microM). Chronic (i.e. 3-h) treatment of cells with (+/-)-bupivacaine increased cell surface [3H]saxitoxin ([3H]STX) binding capacity by 48% (EC(50) of 233 microM; t(1/2)=7.4 h), without changing the K(d) value. Treatment for 24 h with either (+)- or (-)-bupivacaine, or (-)-ropivacaine elevated [3H]STX binding, whereas 24-h treatment with lidocaine had no effect. The rise of [3H]STX binding by (+/-)-bupivacaine was prevented by cycloheximide, an inhibitor of protein synthesis, or brefeldin A, an inhibitor of cell surface vesicular exit from the trans-Golgi network; however, (+/-)-bupivacaine did not increase Na(+) channel alpha- and beta(1)-subunit mRNA levels. In cells subjected to (+/-)-bupivacaine treatment (1 mM for 24 h) followed by 3-h washout, veratridine-induced 22Na(+) influx was enhanced, even when measured in the presence of ouabain, an inhibitor of Na(+),K(+)-ATPase. Ptychodiscus brevis toxin-3 potentiated veratridine-induced 22Na(+) influx by 2.3-fold in the (+/-)-bupivacaine-treated cells, as in non-treated cells. These results suggest that lipophilic bupivacaine enantiomers or (-)-ropivacaine acutely inhibit Na(+) channel gating, whereas its chronic treatment up-regulates cell surface expression of Na(+) channels via translational and externalization events.

To test whether acute volume expansion can normalize orthostatic intolerance and autonomic tone after prolonged bed rest (BR), 23 men were subjected to 20 days BR. Left ventricular (LV) echocardiography was performed during the lower body negative pressure (LBNP) test before and after BR with and without preceding rapid infusion of saline (1,500 ml/30 min). Saline infusion restored heart rate, LV dimension, and stroke volume during LBNP, increased cardiac output (from 4.1 +/- 1 to 5.3 +/- 1 L/min), and normalized LBNP tolerance time (from 11 +/- 4 to 23 +/- 6 minutes). In 9 men, a Holter electrocardiogram was recorded on the day before BR, the fourth and twentieth days of BR, and the day after BR. The high-frequency component of heart rate variability during sleep gradually decreased and reached the lowest level on the day after BR (100%, 66 +/- 16%, 39 +/- 18%, 10 +/- 8%). Thus, restoring decreased blood volume is an effective countermeasure for orthostatic intolerance after BR. However, decreased vagal tone persisted, suggesting reset autonomic tone.

The central nervous system requires an effective autoregulation of cerebral circulation in order to meet the critical and unusual demands of the brain. In addition, cerebral microvessels has a unique feature, the formation of the blood-brain barrier, which contributes to the stability of the brain parenchymal microenvironment. Many factors are known to be involved in the regulation of cerebral circulation and blood-brain barrier functions. In the last few years a new potential candidate, adrenomedullin, a hypotensive peptide was added to this list. Adrenomedullin has a potent vasodilator effect on the cerebral vasculature, and it may be implicated in the pathologic mechanism of cerebrovascular diseases. In this review, we describe current knowledge about the origin and possible role of adrenomedullin in the regulation of cerebral circulation and blood-brain barrier functions.

The middle ear cavity is an air-filled space that must be maintained for effective sound transmission to the inner ear. To examine the mechanisms of water homeostasis in the middle ear, we investigated whether aquaporins (AQPs), a family of water-permeable channels, were expressed in the middle ear. Reverse transcription-polymerase chain reaction and immunoblot analyses revealed that mRNAs encoding AQP1, 4 and 5 (but not 2 or 3) subtypes were expressed in rat middle ear epithelium; AQP1, 4 and 5 were detected as 28-, 30- and 30-kDa proteins, respectively. Immunohistochemical analysis showed that AQP1 was localized at capillary endothelial cells and fibroblasts in lamina propria mucosae; AQP4 was present solely at the basolateral membrane of ciliated cells, whereas AQP5 was on the apical surface of ciliated cells as well as of flat and columnar epithelial cells. The characteristic different localizations of AQP1, 4 and 5 subtypes in the middle ear suggest that middle ear water homeostasis requires the coordinated operation of these AQPs.

Long-term (or =12 h) treatment of cultured bovine adrenal chromaffin cells with thapsigargin (TG), an inhibitor of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), caused a time (t(1/2)=16.3 h)- and concentration (IC50=37.8 nM)-dependent decrease of cell surface 125I-insulin binding by 35%, but did not change the Kd value. TG caused a sustained increase of cytoplasmic concentration of Ca2+ ([Ca2+]c) in a biphasic manner, and the effect of TG on 125I-insulin binding was abolished by BAPTA-AM. Western blot analysis showed that TG lowered insulin receptor (IR) beta-subunit level in membrane, but did not alter total cellular levels of IR precursor and IR beta-subunit. Internalization of cell surface IR, as measured by using brefeldin A, an inhibitor of vesicular exit from the trans-Golgi network (TGN), was not changed by TG. These results suggest that inhibition of SERCA by TG and the subsequent increase of [Ca2+]c down-regulates cell surface IR by retarding externalization of IR from the TGN.

We studied whether a novel proadrenomedullin derived peptide was present and what was its physiological function in cultured bovine adrenal chromaffin cells. We found a high level of proadrenomedullin N-terminal 12 peptide (PAMP-12) which consists of a peptide from 9th amino acid to 20th amino acid of proadrenomedullin N-terminal 20 peptide (PAMP-20). PAMP-12 was released from the cells along with catecholamine upon stimulation of nicotinic cholinergic receptors. When PAMP-12 was added in the incubation medium, this peptide inhibited nicotinic receptor-mediated catecholamine release and influx of Na + and Ca 2+ into the cells. PAMP-12 did not affect catecholamine release evoked by histamine or by depolarization by high concentration of potassium. PAMP-12 also inhibited synthesis of catecholamines as well as the activation of tyrosine hydroxylase by nicotinic stimulation. Thus, PAMP-12 is an endogenous peptide that regulates release and synthesis of catecholamines by acting on nicotinic cholinergic receptors in an autocrine manner in adrenal chromaffin cells.

Long-term (>/=3 h) treatment of cultured bovine adrenal chromaffin cells with cyclosporin A (CsA) decreased cell surface (125)I-insulin binding by 62% in a concentration (IC(50)=18 microM)- and time (t(1/2)=16 h)-dependent manner, but did not change the K(d) value. FK506 (1 microM) or rapamycin (3 microM) treatment reduced (125)I-insulin binding. Western blot analysis showed that CsA treatment decreased insulin receptor (IR) beta-subunit level (t(1/2)=15 h) in membrane fraction, but did not alter total cellular levels of IR precursor and IR beta-subunit. Internalization rate of cell surface IR measured by using brefeldin A, an inhibitor of vesicular exit from the trans-Golgi network, was comparable between non-treated and CsA-treated cells. Thus, CsA, FK506 and rapamycin inhibit peptidyl prolyl cis-trans isomerase activities of cyclophilin and FK506-binding protein, and down-regulate IR presumably by reducing cell surface externalization of IR.

To identify alpha(1)-adrenergic receptor subtypes involved in the regulation of cerebral microcirculation, we studied alpha(1)-adrenergic receptor subtypes expressed in the rat cerebral microvessels. The microvessels were prepared from rat cerebral cortex by albumin flotation and glass bead filtration techniques. [(125)I]HEAT binding to the cerebral microvessels was displaced by low concentrations of 5-methylurapidil, a selective antagonist for alpha(1A)-receptors, and modified Scatchard analysis of the data revealed that half of alpha(1)-receptors is alpha(1A)-subtype, and that alpha(1B)- and/or alpha(1D)-receptors are also present. The K(i) value of the high-affinity component for 5-methylurapidil was 3.90+/-1.08 nM, which is comparable with the value obtained in the rat cerebral cortex (2.17+/-0.88 nM). Reverse transcription and polymerase chain reaction experiments showed that mRNAs of alpha(1A)- and alpha(1B)-receptors, but not alpha(1D)-receptors, were expressed in the cerebral microvessels. These results suggest that alpha(1)-receptors involved in the regulation of cerebral microvessel function are alpha(1A)- and alpha(1B)-receptor subtypes.

In cultured bovine adrenal chromaffin cells, NS-7 [4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy) pyrimidine hydrochloride], a newly-synthesized neuroprotective drug, inhibited nicotine-induced 22 Na + influx via nicotinic receptors (IC 50 =15.5 μM); the suppression by NS-7 was observed in the presence of ouabain, an inhibitor of Na + ,K + -ATPase, and was not attenuated upon the washout of NS-7. NS-7 decreased nicotine-induced maximum influx of 22 Na + without altering the EC 50 value of nicotine. Also, NS-7 diminished nicotine-induced 45 Ca 2+ influx via nicotinic receptors and voltage-dependent Ca 2+ channels (IC 50 =14.1 μM) and catecholamine secretion (IC 50 =19.5 μM). These results suggest that NS-7 produces noncompetitive and long-lasting inhibitory effects on neuronal nicotinic receptors in adrenal chromaffin cells, and interferes with the stimulus-secretion coupling.

Xylanases hydrolyse the beta-1,4-glycosidic bonds within the xylan backbone and belong to either family 10 or 11 of the glycoside hydrolases, on the basis of the amino acid sequence similarities of their catalytic domains. Generally, xylanases have a core catalytic domain, an N and/or C-terminal substrate-binding domain and a linker region. Until now, X-ray structural analyses of family 10 xylanases have been reported only for their catalytic domains and do not contain substrate-binding domains. We have determined the crystal structure of a family 10 xylanase containing the xylan-binding domain (XBD) from Streptomyces olivaceoviridis E-86 at 1.9 A resolution. The catalytic domain comprises a (beta/alpha)(8)-barrel topologically identical to other family 10 xylanases. XBD has three similar subdomains, as suggested from a triple-repeat sequence, which are assembled against one another around a pseudo-3-fold axis, forming a galactose-binding lectin fold similar to ricin B-chain. The Gly/Pro-rich linker region connecting the catalytic domain and XBD is not visible in the electron density map, probably because of its flexibility. The interface of the two domains in the crystal is hydrophilic, where five direct hydrogen bonds and water-mediated hydrogen bonds exist. The sugar-binding residues seen in ricin/lactose complex are spatially conserved among the three subdomains in XBD, suggesting that all of the subdomains in XBD have the capacity to bind sugars. The flexible linker region enables the two domains to move independently and may provide a triple chance of substrate capturing and catalysis. The structure reported here represents an example where the metabolic enzyme uses a ricin-type lectin motif for capturing the insoluble substrate and promoting catalysis.

In order to determine the gene structure and promoter region of vacuolar H + -translocating inorganic pyrophosphatase (V-PPase), we isolated the genomic clones using a rice BAC library and probes derived from rice V-PPase cDNA ( OVP1 ). The entire OVP1 gene is approx. 5.4 kb in length, and seven introns interrupt the coding sequence of OVP1 . The first intron is extremely large (1869 bp), while the other introns are between 82 and 170 bp. A transcription initiation site, identified by a primer extension analysis, indicated the first exon to be 366 bp. A 1.1 kb fragment containing the 5′-flanking region of the first exon with the GUS reporter gene showed specific promoter activity in rice cells. These data show that the OVP1 gene is composed of eight exons and seven introns, and regulatory elements are present within 1.1 kb upstream from the first exon.

Atrial, brain, and C-type natriuretic peptides of the atrial natriuretic peptide family are present in adrenal chromaffin cells, and are secreted with catecholamines by exocytosis. These peptides modulate the physiological functions of the cells such as synthesis and secretion of catecholamines in an autocrine manner interacting with natriuretic peptide receptors.

Using a simple long-range inverse PCR method, we cloned the GC-rich gene (68%) for an F 10 xylanase from Streptomyces olivaceoviridis E-86. The open reading frame of the cloned gene, fxyn, contained 1431 bp and encoded 477 amino acid residues. FXYN resembled a xylanase of the F 10 family and had two functional domains (a catalytic domain and a substrate-binding domain). Unique triple repeat sequence regions (CLD-C) were located in the substrate-binding domain, which was similar to the xylan-binding domains of xylanase A and that of arabinofuranosidase B from S. lividans. FXYN with a tag that consisted of six histidine residues at the carboxy-terminus was expressed at high levels in Escherichia coli and had the same properties as the native xylanase produced by S. olivaceoviridis. Moreover, the xylan-binding domain of FXYN significantly enhanced hydrolysis of insoluble xylan whereas it had minimal effect on the hydrolysis of soluble xylan.

An intracellular alginate lyase was purified from Enterobacter cloacae M-1 by successive fractionation on Q Sepharose FF, SP Sepharose FF, and Sephacryl S-200 HR. The purified enzyme gave a single band on SDS-PAGE and isoelectric focusing. The enzyme easily degraded polyguluronate and produced unsaturated oligoguluronic acids with a wide range of dp. The major end product of the enzyme reaction on polyguluronate was unsaturated triuronic acid. The pattern of oligoguluronic acids (dp 2–9) generated with the enzyme was investigated by fluorophore-assisted carbohydrate electrophoresis. The enzyme was not capable of degrading oligoguluronic acids having a dp < 4. The degradation rate of heptaguluronic acid by this enzyme remarkably increased, compared with that of hexaguluronic acid, and heptaguluronic acid had a single preferential point of cleavage by this enzyme. On the basis of the cleavage pattern of oligoguluronic acids, the number of subsites was estimated to be seven for this enzyme. The catalytic site of the enzyme is located between the second and the third subsites from the non-reducing end.

We investigated the effect of recombinant human interleukin-1 beta (IL-1 beta) on catecholamine secretion from cultured bovine adrenal medullary cells. Treatment of cultured cells with IL-1 beta (10 ng/ml) for 24 hr caused an increase in accumulation of catecholamines in the cultured medium. The accumulation of catecholamines stimulated by IL-1 beta was observed in time (4-48 hr)- and concentration (3-30 ng/ml)-dependent manners. The stimulatory effect of IL-1 beta (10 ng/ml) was completely inhibited by recombinant human IL-1 receptor antagonist (1 microgram/ml). IL-1 beta had little effect on [3H]norepinephrine uptake to cultured cells. These results suggest that IL-1 beta stimulates catecholamine secretion through activation of IL-1 receptors in adrenal medullary cells.

To characterize sites of action of C-type natriuretic peptide (CNP) in the glial cells, the effect of CNP on cGMP accumulation and the binding of [125I]CNP in rat astrocyte RCR-1 cells were studied. CNP stimulated cGMP accumulation in the cells from 10(-9) M in a dose-dependent manner, but ANP (atrial natriuretic peptide) had a negligible effect on cGMP accumulation in the cells. [125I]CNP was bound to the cells and its Kd value was 2 orders of magnitude lower than that of the ED50 value for stimulation of cGMP accumulation in the cells. Not only CNP but also ANP displaced [125I]CNP binding to the cells. These results suggest that RCR-1 cells have a B-receptor which contains a guanylate cyclase domain and is preferentially activated by CNP, and that they also have a C-receptor which does not contain a guanylate cyclase domain that reacts with both ANP and CNP.

The effect of cell contact on the expression of cell adhesion molecule L1 was investigated. The rat pheochromocytoma cell line PC12 cells were cultured at various densities in the presence or absence of the nerve growth factor. The addition of the nerve growth factor promoted the expression of L1. The expression of L1 was higher when the cells were cultured at high density than when done at low density both in the presence or absence of the nerve growth factor. Immunohistochemical staining of L1 showed that the expression of L1 was higher in the cells contacting each other. These results show that cell interaction affects the expression of cell adhesion molecule L1 in the PC12 cells.

We used El mice, a ddY mouse-derived, autosomal mutant strain and a model of hereditary sensory-precipitated epilepsy, to test the hypothesis that epileptic susceptibility may be associated with the activity of voltage-dependent ion channels. We examined the saxitoxin binding capacity of the receptor site 1 of the Na+ channel α-subunit, the expression activity of the Na+ channel mRNA, the veratridine-induced22Na+ influx in the brain synaptosomes, and the regional distribution of Na+ channels in the brain. Compared with control ddY mice, in El mice which have not experienced seizures, the number of Na+ channels in the brain synaptosomes increased by approximately 20% starting at the fourth postnatal week through the adult stage as determined by [3H]saxitoxin binding assay. Northern blot hybridization analysis showed excess expression of Na+ channel mRNA (by 30–40%) coincidentally with Na+ channel increases. Regional analysis using the saxitoxin binding assay demonstrated approximately 1.3-fold denser distribution of Na+ channels in the cortex and cerebellum but not the hippocampus and midbrain including thalamus of El mice compared to ddY mice. Scatchard plot analysis for saxitoxin binding in the cortex of El mouse brains revealed higher maximum binding capacity (Bmax) values (ddY, 4.43 ± 0.28pmol/mg protein; El,5.43 ± 0.25pmol/mg protein) without a change in Kd (ddY, 1.05 ± 0.03nM; El,1.03 ± 0.01nM). Lastly, veratridine-evoked22Na+ influx, sensitive to tetrodotoxin, was increased approximately 45% in the cortical synaptosomes in six-week-old El mice. These results suggest that El brains characteristically produce an excess of voltage-dependent Na+ channels which is responsible for hyperexcitability of the central nervous system and may account for the predisposition of El mice to epileptic seizures.

Expression of the neural cell adhesion molecule was compared in endplate and non-endplate regions of skeletal muscles of mature and old CBF-1 mice, in order to determine whether age-related changes in neuromuscular morphology were correlated with age changes in neural cell adhesion molecule expression. Three muscles were examined: two (soleus and sternomastoid) showed age-related regionalization of nerve terminals as one manifestation of increased synaptic remodelling while the third (diaphragm) did not. Relative neural cell adhesion molecule content in these muscles was measured by densitometry of immunoblots after concentration by affinity chromatography. Expression of the major 140,000 mol. wt form of neural cell adhesion molecule, which was most abundant in the endplate region, was increased in sternomastoid and soleus of old compared to adult mouse, but was unchanged with age in diaphragm. A 70,000-80,000 mol. wt presumably proteolytic polypeptide fragment of neural cell adhesion molecule was increased in immunoblots of all old muscles. Immunocytochemical studies of skeletal muscles showed no difference in neural cell adhesion molecule cellular distribution in mature vs old mice, but in motor nerve of sternomastoid, the number of neural cell adhesion molecule-positive nerve fibers was increased in old mice. Several lines of evidence indicated that partial denervation was rare in old CBF-1 mice, and therefore could not account for the findings above. Selective increase of 140,000 mol. wt neural cell adhesion molecule expression in the junctional regions of those muscles of old mice which show neuromuscular remodelling indicates that this adhesion molecule may play a role in the age-related instability of motor nerve terminals.

This paper tests for a unit root in Japanese GNP between 1955 and 1987, paying particular attention to the kink in the GNP time series. A careful examination of the kinked point shows that Japanese GNP has a unit root in samples both before and after the kinked point, in contrast to Takeuchi's (1987) research, which specified the kinked point a priori. In addition, fluctuations in GNP are caused mainly by permanent shocks; transitory shocks play a minor role.

In cultured bovine adrenal medullary cells, proadrenomedullin N-terminal 20 peptide (PAMP), at concentrations > or = 3 microM, inhibited carbachol-induced [14C]catecholamine synthesis from [14C]tyrosine. Carbachol-induced activation of tyrosine hydroxylase was also attenuated by PAMP. These results suggest that PAMP is a novel endogenous peptide that regulates catecholamine synthesis via the suppression of its rate-limiting enzyme in adrenal medullary cells.

Effect of CNP on cGMP level in cultured rat cerebral microvessels was investigated. The cerebral microvessels were prepared from rat cerebral cortex by dispase and collagenase digestion and Percoll gradient centrifugation, and cultured. CNP increased cGMP level in a dose-dependent manner suggesting that CNP has a regulatory role in the cerebral microvessel function.

Effect of PACAP on cAMP level in the rat cerebral microvessels was investigated. The cerebral microvessels were prepared from rat cerebral cortex by albumin flotation and glass beads filtration technique. When the microvessels were incubated with PACAP 27, PACAP 38 and VIP, cAMP in the microvessels was increased rapidly reaching a plateau value within 60 s. PACAP 27, PACAP 38 and VIP increased cAMP level in a dose-dependent manner with EC50 values of 4.7, 7.0 and 34 nM, respectively. These results suggest that PACAPs play a role in the regulation of the cerebral microvessel function.

Subtypes of protein kinase C were analyzed in adrenal and extra-adrenal pheochromocytoma of humans. Almost all protein kinase C of the adrenal tumor was type III, while the enzyme of the extra-adrenal tumor was separated into two major fractions corresponding to type II and type III by hydroxyapatite column chromatography. The extra-adrenal tumor but not the adrenal tumor spontaneously produced neurite-like processes when the cells were cultured in vitro. These results suggest that the high proportion of type II enzyme may reflect neuron-directed differentiation in human pheochromocytoma.

Subtypes of protein kinase C were determined in tissues derived from neural crest. The bovine adrenal medulla and rat superior cervical ganglia contained type III enzyme as the major subtype with a small amount of type II enzyme. In PC12 cells, treated with or without nerve growth factor, type III was the major subtype but a minor peak which is distinct from types II and III was observed. These results show that the type III enzyme is prevalent in various differentiation stages of neural crest and suggest that the enzyme relevant to the regulation of catecholamine synthesis and release in these cells is type III.