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The Eastern Iratsu body of the Sanbagawa metamorphic terrane in central Shikoku recorded an Early Jurassic metamorphic event older than the Sanbagawa metamorphic event. This study carried out the petrochronology of eclogitic high-pressure marble in the Eastern Iratsu body to determine the corresponding conditions of metamorphism and their variation in time. A combination of trace element (Zr) thermometry, host-inclusion mineral elastic barometry, and in-situ U–Pb dating was performed on the titanite in an eclogitic marble to reveal the metamorphic pressure/temperature conditions of 2.3–2.7 GPa and 1000–1200 °C high-temperature (HT) eclogite facies metamorphic conditions, which occurred at approximately 200 Ma. Moreover, certain titanite grains exhibited a distinctly younger age of 164 Ma with a similar Zr concentration. The 164 Ma domain of titanite is observed near the Na-enriched diopside, indicating the fluid-infiltration and corresponding titanite recrystallization. Some diopside in the matrix contains high-salinity aqueous fluid inclusions (freezing point depression = 3.6–3.7 °C) in the Na-enriched domain, which may infer the source of Na. Furthermore, titanite rims containing trace amounts of Zr yielded 126 Ma, which is comparable with that of the early Sanbagawa metamorphism; however, no P-T estimation was available. This newly identified Early Jurassic metamorphic event is much older than the previously reported metamorphic age of the Sanbagawa metamorphic belt, i.e., c. 120 Ma for early Sanbagawa metamorphism. Although the origin of the HP marble is unclear, the pre-Sanbagawa HT eclogite facies metamorphism reported here could provide a new insight for the subduction of the Izanagi plate and the corresponding geotectonic evolution of eastern Asia.

PURPOSE Common labs such as a daily complete blood count or a daily basic metabolic panel represent possible waste and have been targeted by professional societies and the Choosing Wisely campaign for critical evaluation. We undertook a multifaceted quality-improvement (QI) intervention in a large community hospitalist group to decrease unnecessary common labs. METHODS The QI intervention was composed of academic detailing, audit and feedback, and transparent reporting of the frequency with which common labs were ordered as daily within the hospitalist group. We performed a pre-post analysis, comparing a cohort of patients during the 10-month baseline period before the QI intervention and the 7-month intervention period. Demographic and clinical data were collected from the electronic medical record. The primary endpoint was number of common labs ordered per patient-day as estimated by a clustered multivariable linear regression model clustering by ordering hospitalist. Secondary endpoints included length of stay, hospital mortality, 30-day readmission, blood transfusion, amount of blood transfused, and laboratory cost per patient. RESULTS The baseline (n = 7824) and intervention (n = 5759) cohorts were similar in their demographics, though the distribution of primary discharge diagnosis-related groups differed. At baseline, a mean of 2.06 (standard deviation 1.40) common labs were ordered per patient-day. Adjusting for age, sex, and principle discharge diagnosis, the number of common labs ordered per patient-day decreased by 0.22 (10.7%) during the intervention period compared to baseline (95% confidence interval [CI], 0.34 to 0.11; P

Signals from intracellular glucocorticoids (GCs) via 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) in adipose tissues have been reported to serve as amplifiers leading to deterioration of glucose metabolism associated with obesity. To elucidate adipose dysfunction via 11β-HSD1 activation in the development of obesity-related diabetes, we established novel diabetic mice by implanting a cortisone pellet (CP) in diet-induced obesity (DIO) mice. Cortisone pellet-implanted DIO mice (DIO/CP mice) showed hyperglycemia, insulin resistance, hyperlipidemia, and ectopic fat accumulation, whereas cortisone pellet implantation in lean mice did not induce hyperglycemia. In DIO/CP mice, indexes of lipolysis such as plasma glycerol and nonesterified fatty acids (NEFAs) increased before hyperglycemia appeared. Furthermore, the adipose mRNA level of 11β-HSD1 was up-regulated in DIO/CP mice compared with sham-operated DIO mice. RU486 (mifepristone, 11β-[p-(dimethylamino)phenyl]-17β-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one), a glucocorticoid receptor antagonist, decreased adipose mRNA levels of 11β-HSD1 as well as adipose triglyceride lipase. RU486 also improved plasma NEFA, glycerol, and glucose levels in DIO/CP mice. These results demonstrate that lipolysis in adipose tissues caused by GC activation via 11β-HSD1 serves as a trigger for diabetes with ectopic fat accumulation. Our findings also indicate the possibility of a vicious circle of GC signals via 11β-HSD1 up-regulation in adipose tissues, contributing to deterioration of glucose metabolism to result in diabetes. Our DIO/CP mouse could be a suitable model of type 2 diabetes to evaluate adipose dysfunction via 11β-HSD1.

Irbesartan, an angiotensin II type 1 receptor blocker has been reported to alleviate metabolic disorder in animal studies and human clinical trials. Although this effect may be related to the ability of irbesartan to serve as a partial agonist for the peroxisome proliferator-activated receptor (PPAR)-γ, the target tissues on which irbesartan acts remain poorly defined. As muscle glucose transport plays a major role in maintaining systemic glucose homeostasis, we investigated the effect of irbesartan on glucose uptake in skeletal muscle cells. In C2C12 myotubes, 24-h treatment with irbesartan significantly promoted both basal and insulin-stimulated glucose transport. In L6-GLUT4myc myoblasts, irbesartan caused a significant increase in glucose transport and GLUT4 translocation to the cell surface in a concentration-dependent manner. Valsartan, another angiotensin II type 1 receptor blocker had no effect on either glucose uptake or GLUT4 translocation, implying that these actions on glucose transport are independent of angiotensin II receptor blockade. Moreover, irbesartan exerted these effects in an additive manner with insulin, but not with acute treatment for 3 h, suggesting that they may require the synthesis of new proteins. Finally, in insulin-resistant Zucker fatty rat, irbesartan (50 mg/kg/day for 3 weeks) significantly ameliorated insulin resistance without increasing weight gain. We conclude that irbesartan has a direct action, which can be additive to insulin, of promoting glucose transport in skeletal muscle. This may be beneficial for ameliorating obesity-related glucose homeostasis derangement.

PTP1B(-/-) mice are resistant to diet-induced obesity due to leptin hypersensitivity and consequent increased energy expenditure. We aimed to determine the cellular mechanisms underlying this metabolic state. AMPK is an important mediator of leptin's metabolic effects. We find that alpha1 and alpha2 AMPK activity are elevated and acetyl-coenzyme A carboxylase activity is decreased in the muscle and brown adipose tissue (BAT) of PTP1B(-/-) mice. The effects of PTP1B deficiency on alpha2, but not alpha1, AMPK activity in BAT and muscle are neuronally mediated, as they are present in neuron- but not muscle-specific PTP1B(-/-) mice. In addition, AMPK activity is decreased in the hypothalamic nuclei of neuronal and whole-body PTP1B(-/-) mice, accompanied by alterations in neuropeptide expression that are indicative of enhanced leptin sensitivity. Furthermore, AMPK target genes regulating mitochondrial biogenesis, fatty acid oxidation, and energy expenditure are induced with PTP1B inhibition, resulting in increased mitochondrial content in BAT and conversion to a more oxidative muscle fiber type. Thus, neuronal PTP1B inhibition results in decreased hypothalamic AMPK activity, isoform-specific AMPK activation in peripheral tissues, and downstream gene expression changes that promote leanness and increased energy expenditure. Therefore, the mechanism by which PTP1B regulates adiposity and leptin sensitivity likely involves the coordinated regulation of AMPK in hypothalamus and peripheral tissues.

We attempted the development of highly efficient fishing lights using LEDs for saury fishing. Considering the spectral luminous efficacy characteristic of a Pacific saury, LED fishing lights were fabricated. Transmission properties in sea water in LED fishing lights were investigated, and it was found that the fabricated LED fishing lights sufficiently can be utilized even in sea water. Furthermore, we attempted the saury-fishing experiments by using the LED fishing lights, and comparable catch of Pacific saury was achieved by approximately 55% electric power by using both LED poles and incandescent lamp poles.

AMP-activated protein kinase (AMPK) is a key regulator of cellular energy balance and of the effects of leptin on food intake and fatty acid oxidation. Obesity is usually associated with resistance to the effects of leptin on food intake and body weight. To determine whether diet-induced obesity (DIO) impairs the AMPK response to leptin in muscle and/or hypothalamus, we fed FVB mice a high fat (55%) diet for 10-12 weeks. Leptin acutely decreased food intake by approximately 30% in chow-fed mice. DIO mice tended to eat less, and leptin had no effect on food intake. Leptin decreased respiratory exchange ratio in chow-fed mice indicating increased fatty acid oxidation. Respiratory exchange ratio was low basally in high fat-fed mice, and leptin had no further effect. Leptin (3 mg/kg intraperitoneally) increased alpha2-AMPK activity 2-fold in muscle in chow-fed mice but not in DIO mice. Leptin decreased acetyl-CoA carboxylase activity 40% in muscle from chow-fed mice. In muscle from DIO mice, acetyl-CoA carboxylase activity was basally low, and leptin had no further effect. In paraventricular, arcuate, and medial hypothalamus of chow-fed mice, leptin inhibited alpha2-AMPK activity but not in DIO mice. In addition, leptin increased STAT3 phosphorylation 2-fold in arcuate of chow-fed mice, but this effect was attenuated because of elevated basal STAT3 phosphorylation in DIO mice. Thus, DIO in FVB mice alters alpha2-AMPK in muscle and hypothalamus and STAT3 in hypothalamus and impairs further effects of leptin on these signaling pathways. Defective responses of AMPK to leptin may contribute to resistance to leptin action on food intake and energy expenditure in obese states.

We collected venous blood samples from 7 steady-state patients with homozygous sickle cell disease under venous oxygen pressure without exposure to air (UnExp-blood) and compared the morphological, oxygen-binding, and sickling properties with those of SS cells in aliquots of the same venous blood samples that were oxygenated in room air or at a PO2 near 180 mmHg (Exp-blood). Results showed that (1) upon deoxygenation under nitrogen, UnExp-blood generated a significantly higher percentage of elongated reversibly sickled cells (RSCs) than did Exp-blood; (2) upon gradual oxygenation of completely deoxygenated sickled cells, RSCs in UnExp-blood converted to discocytes at a higher oxygen pressure than did those in Exp-blood; (3) the degree of hysteresis between the sickling/desickling curves of UnExp-blood was greater than that of Exp-blood; and (4) deoxy-Hb S in hemolysate prepared from SS cells in UnExp-blood polymerized without a delay time, while those from Exp-blood polymerized with a distinct delay time. The in vivo properties of RSCs significantly changed upon oxygenation. We also found that the various properties of blood samples collected from patients with SCD by the ordinary method were similar to those of Exp-blood, probably because such blood samples are exposed to oxygen through air in the needle, syringe, and Vacutainer. Once SS cells were oxygenated, the in vivo properties of RSCs could not be recovered by partial deoxygenation to venous oxygen pressure. Am. J. Hematol. 81:26–35, 2006. © 2005 Wiley-Liss, Inc.

15-Deoxy-Delta12,14-prostaglandin J2 (15d-Delta12,14-PGJ2) is an endogenous ligand for a nuclear peroxysome proliferator activated receptor-gamma (PPAR). We found novel binding sites of 15d-Delta12,14-PGJ2 in the neuronal plasma membranes of the cerebral cortex. The binding sites of [3H]15d-Delta12,14-PGJ2 were displaced by 15d-Delta12,14-PGJ2 with a half-maximal concentration of 1.6 microM. PGD2 and its metabolites also inhibited the binding of [3H]15d-Delta12,14-PGJ2. Affinities for the novel binding sites were 15d-Delta12,14-PGJ2 > Delta12-PGJ2 > PGJ2 > PGD2. Other eicosanoids and PPAR agonists did not alter the binding of [3H]15d-Delta12,14-PGJ2. In primary cultures of rat cortical neurons, we examined the pathophysiologic roles of the novel binding sites. 15d-Delta12,14-PGJ2 triggered neuronal cell death in a concentration-dependent manner, with a half-maximal concentration of 1.1 microM. The neurotoxic potency of PGD2 and its metabolites was also 15d-Delta12,14-PGJ2 > Delta12-PGJ2 > PGJ2 > PGD2. The morphologic and ultrastructural characteristics of 15d-Delta12,14-PGJ2-induced neuronal cell death were apoptotic, as evidenced by condensed chromatin and fragmented DNA. On the other hand, we detected little neurotoxicity of other eicosanoids and PPAR agonists. In conclusion, we demonstrated that novel binding sites of 15d-Delta12,14-PGJ2 exist in the plasma membrane. The present study suggests that the novel binding sites might be involved in 15d-Delta12,14-PGJ2-induced neuronal apoptosis.

Mammalian group IIA secretory phospholipase A2 (sPLA2-IIA) generates prostaglandin D2 (PGD2) and triggers apoptosis in cortical neurons. However, mechanisms of PGD2 generation and apoptosis have not yet been established. Therefore, we examined how second messengers are involved in the sPLA2-IIA-induced neuronal apoptosis in primary cultures of rat cortical neurons. sPLA2-IIA potentiated a marked influx of Ca2+ into neurons before apoptosis. A calcium chelator and a blocker of the L-type voltage-sensitive Ca2+ channel (L-VSCC) prevented neurons from sPLA2-IIA-induced neuronal cell death in a concentration-dependent manner. Furthermore, the L-VSCC blocker ameliorated sPLA2-IIA-induced morphologic alterations and apoptotic features such as condensed chromatin and fragmented DNA. Other blockers of VSCCs such as N type and P/Q types did not affect the neurotoxicity of sPLA2-IIA. Blockers of L-VSCC significantly suppressed sPLA2-IIA-enhanced Ca2+ influx into neurons. Moreover, reactive oxygen species (ROS) were generated prior to apoptosis. Radical scavengers reduced not only ROS generation, but also the sPLA2-IIA-induced Ca2+ influx and apoptosis. In conclusion, we demonstrated that sPLA2-IIA potentiates the influx of Ca2+ into neurons via L-VSCC. Furthermore, the present study suggested that eicosanoids and ROS generated during arachidonic acid oxidative metabolism are involved in sPLA2-IIA-induced apoptosis in cooperation with Ca2+.

Cerebrospinal fluid prostaglandin E(2) (PGE(2)) levels are elevated in patients with Alzheimer's disease (AD), suggesting an involvement of PGE(2) in the neurodegeneration. AD is characterized by deposits of amyloid beta protein (Abeta) in various regions of the brain, e.g. the cerebral cortex. In the present study, we investigated the effects of PGE(2) on neuronal survival in primary cultures of rat cortical neurons. PGE(2) had no effect on neuronal cell viability or its morphology. Therefore, we examined the synergistic effects of PGE(2) with Abeta, a neurotoxin. Abeta caused neuronal cell death via apoptosis. PGE(2) significantly suppressed Abeta neurotoxicity, but did not promote the neurotoxicity. Furthermore, PGE(2) ameliorated Abeta-induced apoptotic features such as the condensation of chromatin and the fragmentation of DNA. Abeta increased the influx of Ca(2+) into neurons before cell death. Nimodipine, an inhibitor of the L-type voltage-sensitive calcium channel (L-VSCC), significantly reduced Abeta-potentiated Ca(2+) uptake. On the other hand, there was no effect on the Abeta-induced Ca(2+) influx by an N-VSCC blocker or P/Q-VSCC blockers. Moreover, the inhibitor of L-VSCC suppressed Abeta-induced neuronal cell death, whereas neither an N-VSCC blocker nor P/Q-VSCC blockers affected the neurotoxicity of Abeta. PGE(2) also suppressed the Abeta-induced Ca(2+) influx in a concentration-dependent manner. This study demonstrated that PGE(2) rescues cortical neurons from Abeta-induced apoptosis by reducing Ca(2+) influx in the primary culture. Furthermore, the present study suggested that the inhibition of L-VSCC contributes to the neuroprotective effect of PGE(2).

Amyloid beta protein (A beta), the central constituent of senile plaques in Alzheimer's disease (AD) brain, is known to exert toxic effects on cultured neurons. The role of the voltage-sensitive Ca2+ channel (VSCC) in beta (25-35) neurotoxicity was examined using rat cultured cortical and hippocampal neurons. When L-type VSCCs were blocked by application of nimodipine, beta (25-35) neurotoxicity was attenuated, whereas application of omega-conotoxin GVIA (omega-CgTX-GVIA) or omega-agatoxin IVA (omega-Aga-IVA), the blocker for N- or P/Q-type VSCCs, had no effects. Whole-cell patch-clamp studies indicated that the Ca2+ current density of beta (25-35)-treated neurons is about twofold higher than that of control neurons. Also, beta (25-35) increased Ca2+ uptake, which was sensitive to nimodipine. The 2', 7'-dichlorofluorescin diacetate assay showed the ability of beta (25-35) to produce reactive oxygen species. Nimodipine had no effect on the level of free radicals. In contrast, vitamin E, a radical scavenger, reduced the level of free radicals, neurotoxicity, and Ca2+ uptake. These results suggest that beta (25-35) generates free radicals, which in turn, increase Ca2+ influx via the L-type VSCC, thereby inducing neurotoxicity.

In primary cultures of rat cortical neurons, group IB secretory phospholipase A(2) (sPLA(2)-IB) induced cell death. In rat cortical membranes, there were high affinity binding sites of [125I]sPLA(2)-IB. The high-affinity binding sites were decreased by sPLA(2)-IB and anti-sPLA(2) receptor immunoglobulin G (anti-sPLA(2)R IgG). Furthermore, anti-sPLA(2)R IgG caused neuronal cell death in a concentration-dependent manner. The present study suggests that sPLA(2)-IB induces neuronal cell death via its high-affinity binding sites.

Group IB secretory phospholipase A2 (sPLA2-IB) mediates cell proliferation, cell migration, hormone release and eicosanoid production via its receptor in peripheral tissues. In the CNS, high-affinity binding sites of sPLA2-IB have been documented. However, it remains obscure whether sPLA2-IB causes biologic or pathologic response in the CNS. To this end, we examined effects of sPLA2-IB on neuronal survival in primary cultures of rat cortical neurons. sPLA2-IB induced neuronal cell death in a concentration-dependent manner. This death was a delayed response requiring a latent time for 6 h; sPLA2-IB-induced neuronal cell death was accompanied with apoptotic blebbing, condensed chromatin, and fragmented DNA, exhibiting apoptotic features. Before cell death, sPLA2-IB liberated arachidonic acid (AA) and generated prostaglandin D2 (PGD2) from neurons. PGD2 and its metabolite, Delta12-PGJ2, exhibited neurotoxicity. Inhibitors of sPLA2 and cyclooxygenase-2 (COX-2) significantly suppressed not only AA release, but also PGD2 generation. These inhibitors significantly prevented neurons from sPLA2-IB-induced neuronal cell death. In conclusion, we demonstrate a novel biological response, apoptosis, of sPLA2-IB in the CNS. Furthermore, the present study suggests that PGD2 metabolites, especially Delta12-PGJ2, might mediate sPLA2-IB-induced apoptosis.

Expression of group IIA secretory phospholipase A2 (sPLA2-IIA) is documented in the cerebral cortex (CTX) after ischemia, suggesting that sPLA2-IIA is associated with neurodegeneration. However, how sPLA2-IIA is involved in the neurodegeneration remains obscure. To clarify the pathologic role of sPLA2-IIA, we examined its neurotoxicity in rats that had the middle cerebral artery occluded and in primary cultures of cortical neurons. After occlusion, sPLA2 activity was increased in the CTX. An sPLA2 inhibitor, indoxam, significantly ameliorated not only the elevated activity of the sPLA2 but also the neurodegeneration in the CTX. The neuroprotective effect of indoxam was observed even when it was administered after occlusion. In primary cultures, sPLA2-IIA caused marked neuronal cell death. Morphologic and ultrastructural characteristics of neuronal cell death by sPLA2-IIA were apoptotic, as evidenced by condensed chromatin and fragmented DNA. Before apoptosis, sPLA2-IIA liberated arachidonic acid (AA) and generated prostaglandin D2 (PGD2), an AA metabolite, from neurons. Indoxam significantly suppressed not only AA release, but also PGD2 generation. Indoxam prevented neurons from sPLA2-IIA-induced neuronal cell death. The neuroprotective effect of indoxam was observed even when it was administered after sPLA2-IIA treatment. Furthermore, a cyclooxygenase-2 inhibitor significantly prevented neurons from sPLA2-IIA-induced PGD2 generation and neuronal cell death. In conclusion, sPLA2-IIA induces neuronal cell death via apoptosis, which might be associated with AA metabolites, especially PGD2. Furthermore, sPLA2 contributes to neurodegeneration in the ischemic brain, highlighting the therapeutic potential of sPLA2-IIA inhibitors for stroke.

Common labs such as a daily complete blood count or a daily basic metabolic panel represent possible waste and have been targeted by professional societies and the Choosing Wisely campaign for critical evaluation. We undertook a multifaceted quality-improvement (QI) intervention in a large community hospitalist group to decrease unnecessary common labs.The QI intervention was composed of academic detailing, audit and feedback, and transparent reporting of the frequency with which common labs were ordered as daily within the hospitalist group. We performed a pre-post analysis, comparing a cohort of patients during the 10-month baseline period before the QI intervention and the 7-month intervention period. Demographic and clinical data were collected from the electronic medical record. The primary endpoint was number of common labs ordered per patient-day as estimated by a clustered multivariable linear regression model clustering by ordering hospitalist. Secondary endpoints included length of stay, hospital mortality, 30-day readmission, blood transfusion, amount of blood transfused, and laboratory cost per patient.The baseline (n = 7824) and intervention (n = 5759) cohorts were similar in their demographics, though the distribution of primary discharge diagnosis-related groups differed. At baseline, a mean of 2.06 (standard deviation 1.40) common labs were ordered per patient-day. Adjusting for age, sex, and principle discharge diagnosis, the number of common labs ordered per patient-day decreased by 0.22 (10.7%) during the intervention period compared to baseline (95% confidence interval [CI], 0.34 to 0.11; P 0.01). There were nonsignificant reductions in hospital mortality in the intervention period compared to baseline (2.2% vs 1.8%, P = 0.1) as well as volume of blood transfused in patients who received a transfusion (127.2 mL decrease; 95% CI, -257.9 to 3.6; P = 0.06). No effect was seen on length of stay or readmission rate. The intervention decreased hospital direct costs by an estimated $16.19 per admission or $151,682 annualized (95% CI, $119,746 to $187,618).Implementation of a multifaceted QI intervention within a community-based hospitalist group was associated with a significant, but modest, decrease in the number of ordered lab tests and hospital costs. No effect was seen on hospital length of stay, mortality, or readmission rate. This intervention suggests that a community-based hospitalist QI intervention focused on daily labs can be effective in safely reducing healthcare waste without compromising quality of care.

Phospholipase A2 (PLA2) is proposed to play a role in the repair of the ruptured membrane after axotomy. In neonatal rats, we examined the effect of Group IIA secretory PLA2 (sPLA2-IIA) on axotomy-induced cell death of motoneurons. sPLA2-IIA significantly induced death of axotomized motoneurons. Indoxam, a specific inhibitor for sPLA2-IIA, protected motoneurons from the sPLA2-IIA-induced deterioration. The present study indicated that sPLA2-IIA possessed neurotoxic effect rather than neuroprotective effect against facial nerve.

In this study, we investigated the effect of alpha-eudesmol, which potently inhibits the presynaptic omega-agatoxin IVA-sensitive (P/Q-type) Ca(2+) channel, on neurogenic inflammation following electrical stimulation of rat trigeminal ganglion. Treatment with alpha-eudesmol (0.1-1 mg/kg. i.v.) dose-dependently attenuated neurogenic vasodilation in facial skin monitored by a laser Doppler flowmetry. In addition, alpha-eudesmol (1 mg/kg. i.v.) significantly decreased dural plasma extravasation in analysis using Evans blue as a plasma marker. On the other hand, alpha-eudesmol (1 mg/kg, i.v.) did not affect mean arterial blood pressure in rats. The calcitonin gene-related peptide (CGRP) and substance P (SP) released from activated sensory nerves have recently been suggested to be associated with the neurogenic inflammation. In this study, we also showed that alpha-eudesmol (0.45-45 microM) concentration-dependently inhibits the depolarization-evoked CGRP and SP release from sensory nerve terminals in spinal cord slices. These results indicate that the anti-neurogenic inflammation action of alpha-eudesmol, which does not affect the cardiovascular system, may be due to its presynaptic inhibition of the neuropeptide release from perivascular trigeminal terminals. We also suggest that the omega-agatoxin IVA-sensitive Ca(2+) channel blocker, alpha-eudesmol, may become useful for the treatment of the neurogenic inflammation in the trigemino-vascular system such as migraine.

omega-Agatoxin IVA-sensitive Ca(2+) channels have been thought to be involved in physiological excitatory amino acid glutamate release and these channels may also contribute to the development of ischemic brain injury. Recently, we demonstrated that alpha-eudesmol from Juniperus virginiana Linn. (Cupressaceae) inhibits potently the presynaptic omega-agatoxin IVA-sensitive Ca(2+) channels. In the present study, we investigated the effects of alpha-eudesmol on brain edema formation and infarct size determined after 24 h of reperfusion following 1 h of middle cerebral artery occlusion in rats. We first found that alpha-eudesmol concentration-dependently inhibited glutamate release from rat brain synaptosomes and that its inhibitory effect was Ca(2+)-dependent. In the middle cerebral artery occlusion study, intracerebroventricular (i.c.v.) treatment with alpha-eudesmol significantly attenuated the post-ischemic increase in brain water content. alpha-Eudesmol also significantly reduced the size of the infarct area determined by triphenyltetrazolium chloride staining after 24 h of reperfusion. Using a microdialysis technique, we further demonstrated that alpha-eudesmol inhibits the elevation of the extracellular concentration of glutamate during ischemia. From these results, we suggest that alpha-eudesmol displays an ability to inhibit exocytotic glutamate release and to attenuate post-ischemic brain injury.

Recently, the omega-agatoxin IVA (omega-Aga-IVA)-sensitive Ca2+ channel has been demonstrated to play an important role in the physiological neurotransmitter release in mammalian nerve terminals. In this study, we demonstrate that alpha-eudesmol from Juniperus virginiana Linn. (Cupressaceae) inhibits omega-Aga-IVA-sensitive Ca2+ channels in rat brain synaptosomes and cerebellar Purkinje cells. Thirty millimolar KCl-induced 45Ca2+ uptake into the synaptosomes was inhibited by omega-Aga-IVA but insensitive to omega-conotoxin GVIA (omega-CTX-GVIA, N-type Ca2+ channel blocker) and nicardipine (L-type Ca2+ channel blocker). We found that alpha-eudesmol concentration-dependently inhibited the above synaptosomal 45Ca2+ uptake with an IC50 value of 2.6 microM. Co-treatment with alpha-eudesmol and omega-Aga-IVA did not cause any additive inhibitory effect against the synaptosomal 45Ca2+ uptake. Using the whole-cell patch clamp electrophysiological technique, we further demonstrated that alpha-eudesmol concentration-dependently inhibited omega-Aga-IVA-sensitive Ca2+ channel currents recorded from Purkinje cells with an IC50 value of 3.6 microM. The current-voltage relationship of the omega-Aga-IVA-sensitive Ca2+ channel currents was not changed by alpha-eudesmol. On the other hand, alpha-eudesmol also displayed an inhibitory effect on N-type Ca2+ channel currents recorded from differentiated NG108-15 cells with an IC50 value of 6.6 microM. However, alpha-eudesmol had little inhibitory effect on L-type Ca2+ channel currents. Thus, the present data indicated that alpha-eudesmol is a potent nonpeptidergic compound which blocks the presynaptic omega-Aga-IVA-sensitive Ca2+ channel with relative selectivity.

Amyloid β protein (A β ), has been reported to be toxic to neurons in vitro. However, the molecular mechanism leading to neuronal death remains unknown. Here we report protective effects of phenothiazines, a class of neuroleptic agent, against A β toxicity in primary cultures of rat cortical neurons and PC12 cells. β (25–35), an active sequence of A β , showed dose-dependent reduction of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide dye (MTT) reductivity, and chlorpromazine (CPZ), promethazine or trifluoperazine restored it at micromolar concentration. The significant increase in Ca 2+ uptake by chronic treatment of β (25–35) was reduced not only by nimodipine but also by CPZ. These results suggest that phenothiazines attenuate β (25–35) toxicity possibly by reducing of Ca 2+ influx through L-type Ca 2+ channels.

Hospitals and health systems should consider four strategies for improving documentation: Develop a short list of the most commonly underdocumented or incorrectly documented clinical conditions at the facility. Develop definitions for each of the conditions on this list. Ask the medical director of each specialty area to educate the clinicians in their group on these definitions. Measure and manage documentation performance.

A previously uncharacterized type of sickled cell was found in venous blood of patients with sickle cell disease when blood was collected without exposure to air and fixed immediately with 1% glutaraldehyde solution equilibrated with 5% oxygen. These cells were either elongated, resembling irreversibly sickled cells (ISCs), or nonelongated, with a raisin-like shape. Both types assumed a normal discoidal shape upon full oxygenation. Since these cells exist only under partially oxygenated conditions, they are described as partially oxygenated sickled cells (POSCs). POSCs are morphologically distinct from partially deoxygenated sickled cells formed during deoxygenation by having rounded edges, while the latter have sharp edges. Transmission electron microscopy of POSCs revealed various amounts of misaligned Hb S polymers. Investigations in vitro demonstrated the formation of POSC-like cells by partial oxygenation of deoxygenated cells. Since POSCs contain intracellular fibers and sickle readily upon deoxygenation, they may have clinical and pathological significance.

AMP-activated protein kinase (AMPK), an evolutionarily conserved serine-threonine kinase that senses cellular energy status, is activated by stress and neurohumoral stimuli. We investigated the mechanisms by which adrenergic signaling alters AMPK activation in vivo. Brown adipose tissue (BAT) is highly enriched in sympathetic innervation, which is critical for regulation of energy homeostasis. We performed unilateral denervation of BAT in wild type (WT) mice to abolish neural input. Six days post-denervation, UCP-1 protein levels and AMPK α2 protein and activity were reduced by 45%. In β(1,2,3)-adrenergic receptor knock-out mice, unilateral denervation led to a 25-45% decrease in AMPK activity, protein expression, and Thr(172) phosphorylation. In contrast, acute α- or β-adrenergic blockade in WT mice resulted in increased AMPK α Thr(172) phosphorylation and AMPK α1 and α2 activity in BAT. But short term blockade of α-adrenergic signaling in β(1,2,3)-adrenergic receptor knock-out mice resulted in decreased AMPK activity in BAT, which strongly correlated with enhanced phosphorylation of AMPK on Ser(485/491), a site associated with inhibition of AMPK activity. Both PKA and AKT inhibitors attenuated AMPK Ser(485/491) phosphorylation resulting from α-adrenergic blockade and prevented decreases in AMPK activity. In vitro mechanistic studies in BAT explants showed that the effects of α-adrenergic blockade appeared to be secondary to inhibition of oxygen consumption. In conclusion, adrenergic pathways regulate AMPK activity in vivo acutely via alterations in Thr(172) phosphorylation and chronically through changes in the α catalytic subunit protein levels. Furthermore, AMPK α Ser(485/491) phosphorylation may be a novel mechanism to inhibit AMPK activity in vivo and alter its biological effects.

Exendin-4, a glucagon-like peptide 1 receptor agonist, is a potent therapeutic xenopeptide hormone for the treatment of type 2 diabetes. In order to further improve in vivo activity, we examined the introduction of sialyl N-acetyllactosamine (sialyl LacNAc) to exendin-4. The glycosylated analogue having sialyl LacNAc at position 28 was found to have improved in vivo activity with prolonged glucose-lowering activity.

Glucagon-like peptide 1 (7-36) amide (GLP-1) has been attracting considerable attention as a therapeutic agent for the treatment of type 2 diabetes. In this study, we applied a glycoengineering strategy to GLP-1 to improve its proteolytic stability and in vivo blood glucose-lowering activity. Glycosylated analogues with N-acetylglucosamine (GlcNAc), N-acetyllactosamine (LacNAc), and alpha2,6-sialyl N-acetyllactosamine (sialyl LacNAc) were prepared by chemoenzymatic approaches. We assessed the receptor binding affinity and cAMP production activity in vitro, the proteolytic resistance against dipeptidyl peptidase-IV (DPP-IV) and neutral endopeptidase (NEP) 24.11, and the blood glucose-lowering activity in diabetic db/db mice. Addition of sialyl LacNAc to GLP-1 greatly improved stability against DPP-IV and NEP 24.11 as compared to the native type. Also, the sialyl LacNAc moiety extended the blood glucose-lowering activity in vivo. Kinetic analysis of the degradation reactions suggested that the sialic acid component played an important role in decreasing the affinity of peptide to DPP-IV. In addition, the stability of GLP-1 against both DPP-IV and NEP24.11 incrementally improved with an increase in the content of sialyl LacNAc in the peptide. The di- and triglycosylated analogues with sialyl LacNAc showed greatly prolonged blood glucose-lowering activity of up to 5 h after administration (100 nmol/kg), although native GLP-1 showed only a brief duration. This study is the first attempt to thoroughly examine the effect of glycosylation on proteolytic resistance by using synthetic glycopeptides having homogeneous glycoforms. This information should be useful for the design of glycosylated analogues of other bioactive peptides as desirable pharmaceuticals.

We determined a trace amount of 14 phenols derived from pesticides by high performance liquid chromatography with an electrochemical detector. Phenols from aqueous and solid samples were enriched through exhaustive steam distillation and XAD-4 resin adsorption. Quantitative determinations were done by reverse-phase high performance liquid chromatography without derivatization. The minimum detectable quantity: phenol, 0.1ng; 2, 4-dichlorophenol, 0.4ng; 2, 4, 6-trichlorophenol, 0.4ng; 2-nitrophenol, 0.4ng. The recovery of phenols from egg plant samples fortified with each of 4-chlorophenol, 4-chloro-2-methylphenol and 2, 4-dichlorophenol at 1ppm ranged from 78 to 102%, while that of 4-nitro-phenol was poor. The recovery of 3-methyl-4-nitrophenol was not determined because other materials interfered.

We have developed a method to determine anilines derived from pesticides by high performance liquid chromatography with an electrochemical detector. Pesticides were hydrolyzed with HCl or KOH to the corresponding anilines, which were isolated from the hydrolysates by steam distillation/solvent extraction. Quantitative determinations were done by reversephase high performance liquid chromatography without derivatization. Anilines in subnanogram quantities were detected with an electrochemical detector. Recovery data were obtained from egg plant samples fortified with fluoroimide, aniliazine, linuron, alachlor or iprodione at 0.4ppm. Average recoveries for fluoroimide, anilazine, linuron, alachlor and iprodione were 100, 85, 68, 27 and 89%, respectively.

ガスクロマトグラフィーによる残留農薬の簡易分析法のための精製法として, 活性炭-フロリジルのミニカラムの有用性について検討した. 試料をメタノールで抽出し, 抽出物をトルエンへ転溶させ, トルエン溶液をミニカラムを用いて精製し, ガスクロマトグラフ (ECDおよびFTD) で定量した.25農薬, 15作物について回収率試験を行なった結果, dimethoate および oxadixyl 以外の農薬はすべて90%以上の回収率を得た. 検出限界は有機リン剤が0.01～0.02ppm, カーバメイトおよび合成ピレスロイドが0.05ppm, 有機塩素剤が0.005ppmであった. 本法は簡便かつ迅速な方法であり, 多くの農薬のモニタリングに広く適用できると思われる.

Ketogenic diets have been used as an approach to weight loss on the basis of the theoretical advantage of a low-carbohydrate, high-fat diet. To evaluate the physiological and metabolic effects of such diets on weight we studied mice consuming a very-low-carbohydrate, ketogenic diet (KD). This diet had profound effects on energy balance and gene expression. C57BL/6 mice animals were fed one of four diets: KD; a commonly used obesogenic high-fat, high-sucrose diet (HF); 66% caloric restriction (CR); and control chow (C). Mice on KD ate the same calories as mice on C and HF, but weight dropped and stabilized at 85% initial weight, similar to CR. This was consistent with increased energy expenditure seen in animals fed KD vs. those on C and CR. Microarray analysis of liver showed a unique pattern of gene expression in KD, with increased expression of genes in fatty acid oxidation pathways and reduction in lipid synthesis pathways. Animals made obese on HF and transitioned to KD lost all excess body weight, improved glucose tolerance, and increased energy expenditure. Analysis of key genes showed similar changes as those seen in lean animals placed directly on KD. Additionally, AMP kinase activity was increased, with a corresponding decrease in ACC activity. These data indicate that KD induces a unique metabolic state congruous with weight loss.

We found various levels of a new type of reversibly sickled cell (RSC) with blunt edges in 44 blood samples obtained from 32 steady-state patients with sickle cell disease (SCD) without exposure to air (UnExp-blood). Because these RSCs could be generated in vitro by partial oxygenation of once-deoxygenated SS cells to venous oxygen pressure, we named them “partially oxygenated sickled cells” (POSCs). These RSCs were classified into elongated and non-elongated RSCs, depending on the ratio of the short axis to long axis. The presence of these cells was previously unknown because the standard blood collection method oxygenates most of the POSCs to discocytes due to oxygen in the air space in the needle, syringe, and blood collection tube (Exp-blood). Although the shape of elongated POSCs is similar to that of irreversibly sickled cells (ISCs), POSCs revert to discocytes upon exposure to air. We found the following: (1) the percentage of total sickled cells (total POSCs + ISCs) in UnExp-blood (29.0 ± 14.5%) was significantly higher than the percentage of sickled cells (mainly ISCs) in Exp-blood (7.3 ± 5.7%); (2) the percentage of sickled cells in UnExp-blood was specific to individual patients during steady state, while it decreased at the onset of a vaso-occlusive event; and (3) the percentage of sickled cells in UnExp-blood varied widely among steady-state patients (4–56%). This new type of RSC may be used as an internal biomarker to evaluate the disease state of individual patients. Am. J. Hematol. 80:249–256, 2005. © 2005 Wiley-Liss, Inc.

Gas6, a product of the growth-arrest-specific gene 6, protects cortical neurons from amyloid beta protein (Abeta)-induced apoptosis. Neuronal apoptosis is also caused by human group IIA secretory phospholipase A(2) (sPLA(2)-IIA), which is expressed in the cerebral cortex after brain ischemia. sPLA(2)-IIA induces Ca(2+) influx via L-type voltage-sensitive calcium channels (L-VSCCs), leading to its neurotoxicity. In the present study, we investigated effects of Gas6 on sPLA(2)-IIA-induced cell death in primary cultures of rat cortical neurons. sPLA(2)-IIA caused neuronal cell death in a concentration- and time-dependent manner. Gas6 significantly prevented neurons from sPLA(2)-IIA-induced cell death. Gas6 suppressed sPLA(2)-IIA-induced apoptotic features such as the condensation of chromatin and the fragmentation of DNA. Prior to cell death, sPLA(2)-IIA increased the influx of Ca(2+) into neurons through L-VSCCs. Gas6 significantly inhibited the sPLA(2)-IIA-induced Ca(2+) influx. The blocker of L-VSCCs also suppressed sPLA(2)-IIA-induced neuronal cell death. The cortical cultures contained few non-neuronal cells, indicating that Gas6 affected the survival of neurons directly, but not indirectly via non-neuronal cells. In conclusion, we demonstrate that Gas6 rescues cortical neurons from sPLA(2)-IIA-induced apoptosis. Furthermore, the present study indicates that inhibition of L-VSCC contributes to the neuroprotective effect of Gas6.

Mammalian group IIA secretory phospholipase A2 (sPLA2-IIA) generates prostaglandin D2 (PGD2) and triggers apoptosis in cortical neurons. However, mechanisms of PGD2 generation and apoptosis have not yet been established. Therefore, we examined how second messengers are involved in the sPLA2-IIA-induced neuronal apoptosis in primary cultures of rat cortical neurons. sPLA2-IIA potentiated a marked influx of Ca2+ into neurons before apoptosis. A calcium chelator and a blocker of the L-type voltage-sensitive Ca2+ channel (L-VSCC) prevented neurons from sPLA2-IIA-induced neuronal cell death in a concentration-dependent manner. Furthermore, the L-VSCC blocker ameliorated sPLA2-IIA-induced morphologic alterations and apoptotic features such as condensed chromatin and fragmented DNA. Other blockers of VSCCs such as N type and P/Q types did not affect the neurotoxicity of sPLA2-IIA. Blockers of L-VSCC significantly suppressed sPLA2-IIA-enhanced Ca2+ influx into neurons. Moreover, reactive oxygen species (ROS) were generated prior to apoptosis. Radical scavengers reduced not only ROS generation, but also the sPLA2-IIA-induced Ca2+ influx and apoptosis. In conclusion, we demonstrated that sPLA2-IIA potentiates the influx of Ca2+ into neurons via L-VSCC. Furthermore, the present study suggested that eicosanoids and ROS generated during arachidonic acid oxidative metabolism are involved in sPLA2-IIA-induced apoptosis in cooperation with Ca2+.

Secretory phospholipase A(2) (sPLA(2)) exhibits neurotoxicity in the central nervous system. There are high-affinity binding sites of the porcine pancreatic group IB sPLA(2) (sPLA(2)-IB) in the brain. sPLA(2)-IB causes neuronal cell death via apoptosis in the rat cerebral cortex. Although apoptosis is triggered by an influx of Ca(2+) into neurons, it has not yet been ascertained whether the Ca(2+) influx is associated with the neurotoxicity of sPLA(2)-IB. We thus examined the possible involvement of Ca(2+) in the neurotoxicity of sPLA(2)-IB in the primary culture of rat cortical neurons. sPLA(2)-IB induced neuronal cell death in a concentration- and time-dependent manner. This death was accompanied by condensed chromatin and fragmented DNA, exhibiting apoptotic features. Before apoptosis, sPLA(2)-IB markedly enhanced the influx of Ca(2+) into neurons. A calcium chelator suppressed neurons from sPLA(2)-IB-induced neuronal cell death in a concentration-dependent manner. An L-type voltage-sensitive Ca(2+) channel (L-VSCC) blocker significantly protected the sPLA(2)-IB-potentiated influx of Ca(2+). On the other hand, blockers of N-VSCC and P/Q-VSCC did not. An L-VSCC blocker protected neurons from sPLA(2)-IB-induced neuronal cell death. In addition, the L-VSCC blocker ameliorated the apoptotic features of sPLA(2)-IB-treated neurons. Neither an N-VSCC blocker nor P/Q-VSCC blockers affected the neurotoxicity of the enzyme. In conclusion, these findings demonstrate that the influx of Ca(2+) into neurons play an important role in the neurotoxicity of sPLA(2)-IB. Furthermore, the present study suggests that L-VSCC contribute to the sPLA(2)-IB-potentiated influx of Ca(2+) into neurons.

Endothelin (ET), a vasoconstrictive peptide, acts as an anti-apoptotic factor, and endothelin receptor B (ET(B) receptor) is associated with neuronal survival in the brain. In the Alzheimer's disease (AD) brain, accumulation of amyloid beta protein (Abeta) is thought to cause neuronal cell death via apoptosis. In the present study, we investigated effects of ET(B) receptor agonists on Abeta-induced neuronal cell death. In primary cultures of rat cortical neurons, Abeta(25-35) caused neuronal cell death in a concentration- and time-dependent manner. Abeta(25-35)-induced neuronal cell death was accompanied by chromatin condensation and DNA fragmentation, exhibiting apoptotic features. ET-3 and IRL-1620, ET(B) receptor agonists, significantly prevented neurons from undergoing Abeta(25-35)-induced cell death. Prior to cell death, Abeta increased concentration of intracellular Ca(2+) ([Ca(2+)](i)). Nimodipine, an L-type voltage-sensitive Ca(2+) channel (L-VSCC) blocker, suppressed the Abeta-induced Ca(2) influx, and attenuated Abeta-induced neuronal apoptosis. On the other hand, omega-conotoxin GIVA, an N-type VSCC blocker and omega-conotoxin MVIIC and omega-agatoxin IVA, P/Q-type VSCC blockers, had no effect. ET-3 and IRL-1620 significantly blocked Abeta(25-35)-induced Ca(2) influx. Furthermore, BQ788, an ET(B) receptor antagonist, inhibited both an anti-apoptotic effect and an L-VSCC-inactivating effect of ET(B) receptor agonists. In conclusion, ET(B) receptor agonists exhibit a protective effect against neurotoxicity of Abeta. Furthermore, these agonists appear to act as anti-apoptotic factors by blocking of L-VSCCs.

Group IB secretory phospholipase A2 (sPLA2-IB) mediates cell proliferation, cell migration, hormone release and eicosanoid production via its receptor in peripheral tissues. In the CNS, high-affinity binding sites of sPLA2-IB have been documented. However, it remains obscure whether sPLA2-IB causes biologic or pathologic response in the CNS. To this end, we examined effects of sPLA2-IB on neuronal survival in primary cultures of rat cortical neurons. sPLA2-IB induced neuronal cell death in a concentration-dependent manner. This death was a delayed response requiring a latent time for 6 h; sPLA2-IB-induced neuronal cell death was accompanied with apoptotic blebbing, condensed chromatin, and fragmented DNA, exhibiting apoptotic features. Before cell death, sPLA2-IB liberated arachidonic acid (AA) and generated prostaglandin D2 (PGD2) from neurons. PGD2 and its metabolite, Delta12-PGJ2, exhibited neurotoxicity. Inhibitors of sPLA2 and cyclooxygenase-2 (COX-2) significantly suppressed not only AA release, but also PGD2 generation. These inhibitors significantly prevented neurons from sPLA2-IB-induced neuronal cell death. In conclusion, we demonstrate a novel biological response, apoptosis, of sPLA2-IB in the CNS. Furthermore, the present study suggests that PGD2 metabolites, especially Delta12-PGJ2, might mediate sPLA2-IB-induced apoptosis.

The synthesis and structure activity relationships of a series of N-pyrimidinyl benzenesulfonamides as ETB selective antagonists are described. N-Isoxazolyl benzenesulfonamide 1a, previously reported, (1) was selected as a lead compound, and isosteric replacement of the isoxazole ring of 1a with a pyrimidine ring led to the discovery of the highly potent ETB selective antagonist 6e with oral bioavailability. Modification of the terminal aldehyde group at the 6-position of the pyrimidine ring was investigated, and malonate 15b and acylhydrazone 16f were found to be equipotent to aldehyde 6e. Compound 6e showed ETB antagonistic activity on in vivo evaluation.

Recently, P/Q-type Ca2+ channels have been shown to be involved in neurotransmission in the central nervous system in mammals. We evaluated the effects of the P/Q-type Ca2+ channel blocker omega-agatoxin IVA (omega-Aga-IVA) on brain edema formation and infarct size determined after 24 h of reperfusion following 1 h of middle cerebral artery (MCA) occlusion in rats. Intracerebroventricular (i.c.v.) treatment with omega-Aga-IVA significantly attenuated the postischemic increase of brain water content. omega-Aga-IVA also significantly reduced the size of the infarct area determined by triphenyltetrazolium chloride staining after 24 h of reperfusion. omega-Aga-IVA (30 pmol, i.c.v.), which exhibited a neuroprotective effect, had no significant effect on the magnitude of intra- and postischemic brain temperature when compared with vehicle-treated rats. This indicates that the postischemic neuroprotective effect of omega-Aga-IVA is produced by a direct and not an indirect effect via hypothermia. These results suggest that P/Q-type Ca2+ channels may be involved in the development of focal ischemic brain injury and that blockers of these channels may be therapeutically useful against ischemic injury.

The effects of U50488, kappa-opioid agonist on P-type Ca2+ channels, were studied. U50488 inhibited depolarization-induced Ca2+ uptake into rat brain synaptosomes, which was sensitive to omega-Agatoxin IVA (omega-AgaIVA; P-type Ca2+ channel blocker) and inhibited P-type Ca2+ channel currents recorded from rat cerebellar Purkinje neurons by the whole-cell patch clamp method. Dynorphin A also inhibited P-type Ca2+ channel currents. The inhibition by U50488 was biphasic; high affinity component (21%, IC50 = 8.9 x 10(-8) M) and low affinity component (79%, IC50 = 1.1 x 10(-5) M). At low concentrations of U50488 (10(-6) M), P-type Ca2+ channel current inhibition was attenuated by norbinartorphimine (nor-BNI), kappa-opioid antagonist, and by dialysis of cells with a pipette solution containing guanosine 5'-O-(2-thiodiphosphate) (GDP-beta S). At high concentrations of U50488 (10(-5) M), P-type Ca2+ channel current inhibition was frequency-dependent. Thus U50488-induced current inhibition is mediated by two mechanisms. Its high affinity component is produced by activation of kappa-opioid receptors, whereas the low affinity component is due to its direct action on the P-type Ca2+ channel.