In clinical practice, the measurement of endogenous serum substances in order to estimate glomerular filtration rate (GFR) is commonly performed, and the serum creatinine level has become the most commonly used serum marker of renal function. However, the measurement of the serum creatinine concentration can sometimes lead to an overestimation of GFR, especially in the elderly. In recent years, it has been suggested that GFR can be predicted based on the serum cystatin C concentrations and that the serum cystatin C concentration is not influenced by gender or age. A recent meta-analysis demonstrated that serum cystatin C is a better marker for GFR than serum creatinine. In clinical practice, it has been suggested that serum cystatin C can optimize early detection for diabetic or hypertensive nephropathy. In addition, the use of serum cystatin C is possibly more appropriate for establishing an appropriate dose adjustment of drugs that are mainly eliminated by the kidney.

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The motility of the growth cone, an intracellular apparatus located at the tip of the axon in developing neurons, is thought to govern axonal path-finding and the construction of neuronal networks. Growth cones contain an actin-rich cytoskeleton, and their dynamics are regulated by a wide variety of actin-binding proteins and motor proteins. In this review, we will focus on the principal functions of these proteins, their mutual interactions in vitro, and their possible roles in the dynamics of nerve cell growth cones.

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In the present study, the polyherbal preparation diabegon, containing 18 plant extracts with hypoglycemic activity, was evaluated for its preventive effect during progression of type 2 diabetes in high-fructose-diet–fed rats. Oral administration of diabegon (100 mg/kg body weight) delayed development of glucose intolerance for 4 weeks in comparison with the diabetic control group, and the effect of diabegon was compared to that of the standard insulin sensitizer drug rosiglitazone. Diabegon treatment also ameliorated the elevation of glycosylated haemoglobin, liver glycogen content, plasma insulin, homeostasis model assessment, free fatty acids, triglycerides, total cholesterol, LDL-cholesterol, and VLDL-cholesterol, whereas it increased HDL-cholesterol after 56 days of treatment (P<0.05). The mechanism of action by which diabegon attenuates insulin resistance and dyslipidemia may be through induction of peroxisome proliferator-activated receptor-γ and lipoprotein lipase activity in peripheral tissues (muscles). Moreover, diabegon administration for 56 days also produced no alteration in liver and kidney function tests, which seems to indicate its non-toxicity during treatment. Our present results suggest that diabegon may be included in diabetes mellitus treatment regimens, as a drug with good antidiabetic actions but no toxic manifestations.

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We have previously shown that oridonin isolated from Rabdosia rubescens augmented apoptosis while inhibiting autophagy within 24 h in HeLa cells. However, the mechanisms between apoptosis and autophagy induced by oridonin in A431 cells are largely unknown. Here, it was found that autophagic level is significantly upregulated when A431 cells are pretreated with manumycin A (Ras specific inhibitor) compared with oridonin alone treatment, whereas cells precultured with GW5074 (Raf inhibitor) or PD98059 (ERK inhibitor) did not exhibit such an effect. Ras, but not Raf or ERK, was engaged in the control of oridonin-induced autophagy. At the same time, manumycin A contributes to oridonin-induced downregulation of Ras protein expression. Treatment with the combination of oridonin and manumycin A downregulated phosphorylation of Akt, downstream of phosphatidylinositol 3-OH kinase (PI3-K). Preincubation with the PI3-K inhibitor wortmannin and Akt inhibitor KP372-1 enhanced oridonin-induced apoptosis, whereas it inhibited oridonin-induced autophagy. However, under oridonin treatment, the expression of Beclin-1, which has autophagy-inducing activity, was reduced, suggesting that Beclin-1 did not participate in the oridonin-induced autophagy. Morphologic observations, DNA fragmentation analysis, and LDH activity-based assay showed that 3-methyladenine (3-MA), an inhibitor of autophagy, increased the apoptotic sensitivity of A431 cells to oridonin. In addition, manumycin A contributed to oridonin-induced decrease of mitochondrial membrane potential (Δψm), consistent with the upregulation of Bax/Bcl-2 ratio. In conclusion, Ras negatively regulated autophagy in oridonin-treated A431 cells, which might be associated with activation of class I PI3-K. Downregulation of Δψm and increasing of the ratio of Bax/Bcl-2 might also be partially responsible for the initiation of the autophagic process.

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Previous studies revealed that Guizhi-Fuling-Capsules (GZFLC), a traditional Chinese medical (Kampo) formulation composed of five kinds of medicinal plants, Cinnamomum cassia BLUME (Cinnamomi Cortex), Paeonia lactiflora PALL. (Peonies Radix), Paeonia suffruticosa ANDREWS (Moutan Cortex), Prunus persica BATSCH (Persicae Semen), and Poria cocos WOLF (Hoelen), exerts a protective effect against vascular injury and has a protective effect against glutamate- or nitro oxide-mediated neuronal damage. In the present study, the effect of GZFLC in a rat in vivo model of focal cerebral ischemia and reperfusion was investigated. Administration of GZFLC (0.3 and 0.9 g/kg, p.o.) after focal cerebral ischemia significantly decreased brain infarction and water contents in rats subjected to 2-h ischemia followed by 24-h reperfusion from 31.72 ± 2.49%, 84.76 ± 1.63% in the model group to 17.31 ± 3.66%, 82.51 ± 1.36% and 8.30 ± 3.73%, 81.35 ± 1.73%, respectively. Furthermore, analysis of inflammatory cytokines in ischemic brain showed that GZFLC treatment significantly down-regulated expressions of pro-inflammatory cytokines including interleukin (IL)-1β and tissue necrosis factor-α and markedly up-regulated expressions of anti-inflammatory cytokines IL-10 and IL-10R both in mRNA and protein levels. The serum levels of these inflammatory cytokines were also regulated the same way. These results suggested that GZFLC may be beneficial for the treatment of brain ischemia-reperfusion injury partly due to its anti-inflammatory properties.

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This study investigated endogenous mediators involved in mosquito allergy-associated itching in mice. An intradermal injection of an extract of mosquito salivary gland elicited marked scratching in sensitized mice. The 5-lipoxygenase inhibitor zileuton (100 mg/kg), the 5-lipoxygenase activating peptide inhibitor MK-886 (10 mg/kg), and the glucocorticoid betamethasone 17-valerate (3 mg/kg) inhibited the scratching. The scratching was not affected by the cyclooxygenase inhibitors indomethacin and ketoprofen, the TP prostanoid receptor antagonist SQ-29548, the leukotriene B₄ antagonist ONO-4057, the cysteinyl leukotriene antagonist pranlucast, the leukotriene D₄ antagonist MK-571, the platelet-activating factor antagonist CV-3988, the nitric oxide synthase inhibitor N^G-nitro-L-arginine methyl ester, the H₂ histamine-receptor antagonist cimetidine, the H₁ histamine-receptor antagonist terfenadine plus cimetidine, and cypoheptadine that blocks the 5-HT_1/2 serotonin receptors. Zileuton (100 mg/kg) inhibited the increased activity of the cutaneous nerve branch induced by an intradermal injection of the extract, suggesting the peripheral action. Zileuton and MK-886 (10 and 100 μM) did not affect high K⁺-induced increase in intracellular Ca²⁺ concentration in cultured dorsal root ganglion neurons. The results suggest that 5-lipoxygenase metabolite(s) other than leukotriene B₄ and cysteinyl leukotrienes are involved in mosquito allergy-associated itching.

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This study explores the effects of the anti-allergic and anti-fibrotic agent tranilast on adjuvant- and streptococcal cell wall-induced arthritis in rats, animal models of rheumatoid arthritis in humans. Tranilast (150 or 300 mg/kg, twice daily) or vehicle only was administered orally to the two arthritis models, from 17 days before sensitization. As a comparative control, methotrexate (0.1 mg/kg, once daily) was given to another group. Tranilast suppressed the increase in foot volumes, paw thicknesses, clinical scores, and histopathological scores of the ankle joints in both models dose-dependently. In addition, the fibrosis indices of the ankles were dramatically decreased by tranilast in both of the models. Compared to the effects of methotrexate, tranilast seemed to work more effectively in the streptococcal cell wall-induced arthritis model than in the adjuvant-induced arthritis model. From these observations, it can be concluded that tranilast suppresses the development of arthritis in multiple models and is potentially a novel therapeutic agent for human rheumatoid arthritis.

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ATP has broad functions as an autocrine/paracrine molecule. The mode of ATP release and its intracellular source, however, are little understood. Here we show that bradykinin via B₂-receptor stimulation induces the extracellular release of ATP via the inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃]-signaling pathway in cultured taenia coli smooth muscle cells. It was found that bradykinin also increased the production of Ins(1,4,5)P₃ and 2-APB-inhibitable [Ca²⁺]_i. The evoked release of ATP was suppressed by the Ca²⁺-channel blockers, nifedipine, and verapamil. Moreover, the extracellular release of ATP was elicited by photoliberation of Ins(1,4,5)P₃. Bradykinin caused a quick and transient accumulation of intracellular ATP from cells treated with 1% perchloric acid solution (PCA), but not with the cell lysis buffer. Peak accumulation was prevented by 2-APB and thapsigargin, but not by nifedipine or verapamil, inhibitors of extracellular release of ATP. These findings suggest that bradykinin elicits the extracellular release of ATP that is mediated by the Ins(1,4,5)P₃-induced Ca²⁺ signaling and, finally, leads to a Ca²⁺-dependent export of ATP from the cells. Furthermore, the bradykinin-induced transient accumulation of ATP in the cells treated with PCA may imply a possible release of ATP from the endoplasmic reticulum.

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The purpose of the present study was to investigate the influence of lack of histamine (HA) on tumor growth and functions of T cells in order further to illustrate the mechanism of immunological tolerance induction by HA. We assessed the phenotype and cytokine production of splenic lymphocytes in syngeneic HA-free (histidine decarboxylase knock-out) (HDC KO) and wild-type mice, inoculated subcutaneously with the LM2 murine breast cancer cell line. Relative quantification of target mRNA was performed with a TaqMan real-time RT-PCR assay. The CD4⁺CD25^high+ Treg cell numbers were significantly smaller in the tumor-bearing KO mice than in the wild type ones measured by flow-cytometry. The expression of forkhead box P3 (Foxp3) decreased significantly and the copies of splenic Tbox-21 (T-bet) transcriptional factor mRNA was higher in HDC KO tumor-bearing mice than those of normal mice. The cytokine levels showed that a smaller number of interleukin-13-producing Th2 cells were elicited compared to interferon-γ-producing Th1 cells in the tumor-bearing HDC KO mice. In conclusion, the present study demonstrates that endogenous histamine stimulates the growth of breast adenocarcinoma tumor implants in mice by suppressing anti-tumor immunity.

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In this study, we clarified the intracellular mechanism of angiotensin II (Ang II) in promoting migration in rat aortic smooth muscle cells (RASMCs). RASMC migration was measured with the Boyden chamber assay, and the result was confirmed with an aortic sprout assay. The activities of kinases were investigated by western blot analysis. Ang II enhanced RASMC migration, which was chemotaxis directed, and induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase 1/2 (ERK1/2), and heat shock protein 27 (Hsp27). Ang II-enhanced cell migration was inhibited by SB203580 (a p38 MAPK inhibitor) and piceatannol (a spleen tyrosine kinase inhibitor), but only partially by PD98059 (an ERK inhibitor) and PP2 (a Src inhibitor). The Ang II-stimulated phosphorylation of p38 MAPK and Hsp27 in RASMCs was inhibited by piceatannol and SB203580. The phosphorylation of ERK1/2 stimulated by Ang II was suppressed by PD98059, piceatannol, and PP2. Ang II increased the sprout outgrowth from aortic rings and this response was attenuated by pretreatment with SB203580, PD98059, PP2, or piceatannol. These results suggest that p38 MAPK contributes to the regulation of the Ang II-induced chemotactic migration of vascular smooth muscle cells, which is mediated by Hsp27 phosphorylation.

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In the present study, we assessed the effect of the ethanolic extract of the seeds of Cassia obtusifolia (COE) on the learning and memory impairments induced by scopolamine or transient bilateral common carotid artery occlusion (2VO). In a study of the cholinergic dysfunction induced by scopolamine, single COE (25, 50, or 100 mg/kg, p.o.) administration significantly attenuated scopolamine-induced cognitive impairments as determined by the passive avoidance and Y-maze tasks (P<0.05) and also reduced escape-latency on the Morris water maze task (P<0.05). In the 2VO study, COE (50 mg/kg, p.o.) significantly reversed 2VO-induced cognitive impairments in mice by the passive avoidance and the Y-maze tasks (P<0.05). Moreover, COE (50 mg/kg, p.o.) also reduced escape-latency and prolonged swimming time in the target quadrant during a probe trial of the Morris water maze task (P<0.05). In an in vitro study, COE was found to inhibit acetylcholinesterase activity in a dose-dependent manner (IC₅₀ value: 81.6 μg/ml). Furthermore, COE also inhibited acetylcholinesterase activity in an ex vivo study. These results suggest that COE attenuates memory impairment induced by scopolamine or 2VO and that these effects are mediated by enhancing the cholinergic nervous system via acetylcholinesterase inhibition.

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Montelukast, a selective reversible cysteinyl leukotriene D₄-receptor (LTD₄ receptor) antagonist, is used in the treatment of asthma. We have investigated alterations in the glutathione (GSH) and activity levels of antioxidative enzymes [superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), and glutathione reductase (GR)] and myeloperoxidase (MPO), as markers of the ulceration process following oral administration of montelukast, lansoprazole, famotidine, and ranitidine, respectively, in rats with indomethacin-induced ulcers. In the present study, we found that 1) montelukast, lansoprazole, famotidine, and ranitidine all reduced the development of indomethacin-induced gastric damage, with this reduction occurring at a greater magnitude for montelukast, famotidine, and lansoprazole than for ranitidine; 2) montelukast and ranitidine both alleviated increases in the activity levels of CAT and GST enzymes resulting from gastric injury; 3) montelukast and ranitidine both ameliorated depressions in the GSH and activity levels of SOD and GR enzymes caused by indomethacin administration; and 4) all doses of montelukast, lansoprazole, and ranitidine decreased amplification of MPO activity resulting from induced gastric injuries. These results suggest that the gastroprotective effects of montelukast on indomethacin-induced ulcerations can be attributed to its ameliorating effect on oxidative damage and MPO activity.

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The mechanism for noradrenaline (NA)-induced increases in intracellular Ca²⁺ concentration ([Ca²⁺]_i) and physiological significance of Na⁺ influx through receptor-operated channels (ROCs) and store-operated channels (SOCs) were studied in Chinese hamster ovary (CHO) cells stably expressing human α_1A-adrenoceptor (α_1A-AR). [Ca²⁺]_i was measured using the Ca²⁺ indicator fura-2. NA (1 μM) elicited transient and subsequent sustained [Ca²⁺]_i increases, which were inhibited by YM-254890 (G_αq/11 inhibitor), U-73122 (phospholipase C (PLC) inhibitor), and bisindolylmaleimide I (protein kinase C (PKC) inhibitor), suggesting their dependence on G_αq/11/PLC/PKC. Both phases were suppressed by extracellular Ca²⁺ removal, SK&F 96365 (inhibitor of SOC and nonselective cation channel type-2 (NSCC-2)), LOE 908 (inhibitor of NSCC-1 and NSCC-2), and La³⁺ (inhibitor of transient receptor potential canonical (TRPC) channel). Reduction of extracellular Na⁺ and pretreatment with KB-R7943, a Na⁺/Ca²⁺ exchanger (NCX) inhibitor, inhibited both phases of [Ca²⁺]_i increases. These results suggest that 1) stimulation of α_1A-AR with NA elicits the transient and sustained increases in [Ca²⁺]_i mediated through NSCC-2 that belongs to a TRPC family; 2) Na⁺ influx through these channels drives NCX in the reverse mode, causing Ca²⁺ influx in exchange for Na⁺ efflux; and 3) the G_αq/11/PLC/PKC-dependent pathway plays an important role in the increases in [Ca²⁺]_i.

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The present study was performed to examine the effect of tandospirone on sleep latency in a new insomnia animal model by placing rats on a grid suspended over water. For investigating the mechanism of tandospirone, the effect of tandospirone on sleep latency was also studied using rats that were depleted with neuronal serotonin (5-HT) after p-chlorophenylalanine administration. Tandospirone caused a shortening of sleep latency dose-dependently, and a significant effect was observed at 20 mg/kg, p.o. or more. A shortening of sleep latency was observed by administration of p-chlorophenylalanine (300 mg/kg, i.p.) for 2 days. On the other hand, tandospirone exerted no potentiating effect on the shortening of sleep latency induced by p-chlorophenylalanine. From these findings, a shortening of sleep latency induced by tandospirone may occur through the pre-synaptic 5-HT_1A receptors in rats.

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Previous in vitro studies have shown that the degradation of [Met⁵]enkephalin-Arg⁶-Phe⁷ during incubation with cerebral membrane preparations is largely prevented by a mixture of three peptidase inhibitors: amastatin, captopril, and phosphoramidon. The present in vivo study shows that the inhibitory effect of [Met⁵]enkephalin-Arg⁶-Phe⁷ administered intra-third-ventricularly on the tail-flick response was increased more than 1000-fold by the intra-third-ventricular pretreatment with three peptidase inhibitors. The antinociceptive effect produced by the [Met⁵]enkephalin-Arg⁶-Phe⁷ in rats pretreated with any combination of two peptidase inhibitors was significantly smaller than that in rats pretreated with three peptidase inhibitors, indicating that any residual single peptidase could inactivate significant amounts of the [Met⁵]enkephalin-Arg⁶-Phe⁷. The present data, together with those obtained from previous studies, clearly show that amastatin-, captopril-, and phosphoramidon-sensitive enzymes play important roles in the inactivation of endogenous opioid peptides, such as [Met⁵]enkephalin, [Met⁵]enkephalin-Arg⁶-Phe⁷, [Met⁵]enkephalin-Arg⁶-Gly⁷-Leu⁸, and dynorphin A (1-8), administered intra-third-ventricularly to rats.

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We have recently reported that nobiletin, a citrus flavonoid, improves impaired memory in olfactory-bulbectomized (OBX) mice, which have been widely utilized as a useful paradigm that shares some major clinical features of Alzheimer’s disease. Here, we examined the effects of nobiletin on OBX-induced cholinergic neurodegeneration in mice. OBX mice showed reduced acetylcholinesterase (AChE) staining and choline acetyltransferase (ChAT) expression in the hippocampus. An 11-day administration of nobiletin rescued OBX-induced decrease in the density of AChE-staining and ChAT expression in the hippocampus. These results suggest that nobiletin rescues OBX-induced cholinergic neurodegeneration, accompanied by improvement of impaired memory in OBX mice.

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