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Single Administration (single + administration)
Selected AbstractsA Post-ischaemic Single Administration of Galanthamine, a Cholinesterase Inhibitor, Improves Learning Ability in RatsJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 9 2000A. I. ILIEV Transient forebrain ischaemia is widely observed in clinical practice. We have examined the effect of a single administration of the Cholinesterase inhibitor galanthamine (2 mgkg,1, i.p.) 25 min after reperfusion in male Sprague-Dawley rats (180 ± 20 g) after a 20-min common carotid artery occlusion. Twenty-four-hours post-ischaemia there was no difference in motor co-ordination or muscle tonus of the rats treated with or without galanthamine as assessed by the rota-rod test. Learning ability was examined using the shuttle-box test, evaluating the latency time and the number of errors for six days in succession. The performance of the ischaemic saline-injected rats was significantly impaired on days 4, 5, 6 (latency time) compared with the non-ischaemic rats and with the ischaemic animals administered galanthamine (P< 0.05). Similar results were obtained when counting the number of errors (failure to cross the cage during conditioned or unconditioned stimulus). The monitoring of body temperature during the first 12-h post-ischaemia did not show any significant difference between the groups. The data showed a beneficial effect of galanthamine on the recovery of learning ability when administered once only post-ischaemia. This suggests a direct effect on the early pathologic mechanisms of CNS damage. Cholinesterase inhibitors may prove useful in the early clinical treatment of ischaemic conditions. [source] Substance induced plasticity in noradrenergic innervation of the paraventricular hypothalamic nucleusEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 2 2003Arthur S. P. Jansen Abstract Single administration of the cytokine interleukin-1, (IL-1), or the psychostimulant amphetamine, enhanced adrenocorticotropin hormone and corticosterone responses to a stress challenge weeks later. This long-lasting hypothalamic-pituitary-adrenal (HPA)-sensitization is paralleled by an increase in electrically evoked release of noradrenaline in the paraventricular hypothalamic nucleus (PVN). We hypothesized that these functional changes may be associated with morphological plasticity of noradrenergic projections to the PVN, a parameter that shows high reproducibility. Specific alterations in relative (nor)adrenergic innervation density were studied by using dopamine-,-hydroxylase (DBH) as a marker. An image analysis system was used to detect changes in the relative DBH innervation density of the PVN. Groups of adult male rats were given IL-1 (10 µg/kg i.p.), amphetamine (5 mg/kg i.p.), or saline. Three weeks later, IL-1 and amphetamine primed rats showed enhanced adrenocorticotropin hormone and corticosterone responses to an amphetamine challenge. In another set of experiments, the relative DBH innervation density was measured in different PVN subnuclei at four rostro-caudal levels. Single administration of either IL-1 or amphetamine causes three weeks later a selective decrease in relative DBH innervation density in those subnuclei of the PVN that contain high numbers of corticotrophin-releasing hormone (CRH) producing neurons: the dorsal parvocellular and medial parvocellular PVN. We conclude that (1) long-lasting sensitization induced by single exposure to IL-1 and amphetamine induces specific pattern of neuroplastic changes in (nor)adrenergic innervation in the PVN and (2) reduction of relative DBH innervation density in CRH-rich areas is associated with paradoxical increase of electrically evoked release of (nor)adrenaline. [source] A novel role of alkaline phosphatase in protection from immunological liver injury in miceLIVER INTERNATIONAL, Issue 1 2002Qiang Xu Abstract:Aims/Background: Little is known about the role of alkaline phosphatase (AP) in liver diseases, except for its elevation in jaundice or cholestasis. Its substrate, endotoxin, is usually elevated in patients as well as animals with liver damage. This study aimed to provide evidence for its new role as protection against immunological liver damage. Methods: Liver injury was induced in mice by delayed-type hypersensitivity to picryl chloride. AP activity was measured using a commercial kit. Results: In acute liver injury, a significant decrease in AP activity in serum was observed but there was an increase in liver tissue. Single administration of cyclophosphamide before sensitization with picryl chloride exacerbated the liver injury, with more serious AP changes, while consecutive use after the sensitization alleviated the injury with a recovery from the changes. When liver injury proceeded for 1 week, both serum and liver showed decreased AP activity. Lipopolysaccharide facilitated alanine transaminase release from levamisole-pretreated but not non-treated hepatocytes from naive mice. However, the release was confirmed from liver slices of mice with liver injury proceeding for 1 week, even without levamisole pretreatment. Conclusion: The development of liver injury may lead to a dysfunction in AP synthesis and release. Levamisole may make normal hepatocytes, like the hepatocytes from liver-injured mice, highly sensitive to lipopolysaccharide through inhibiting AP synthesis. The findings obtained in this study suggest that AP may contribute to protection from injury by a mechanism involving neutralization of endotoxin. [source] Retinoic acid affects craniofacial patterning by changing Fgf8 expression in the pharyngeal ectodermDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 9 2008Makoto Abe Retinoic acid signaling plays important roles in establishing normal patterning and cellular differentiation during embryonic development. In this study, we show that single administration of retinoic acid at embryonic day 8.5 causes homeotic transformation of the lower jaw into upper jaw-like structures. This homeosis was preceded by downregulation of Fgf8 and Sprouty expression in the proximal domain of the first pharyngeal arch. Downregulation of mesenchymal genes such as Dlx5, Hand2, Tbx1 and Pitx2 was also observed. The oropharynx in retinoic acid-treated embryos was severely constricted. Consistent with this observation, Patched expression in the arch endoderm and mesenchyme was downregulated. Thus, retinoic acid affects the expression of subsets of epithelial and mesenchymal genes, possibly disrupting the regional identity of the pharyngeal arch. [source] Effect of a nutritional liquid supplement designed for the patient with diabetes mellitus (Glucerna SR) on the postprandial glucose state, insulin secretion and insulin sensitivity in healthy subjectsDIABETES OBESITY & METABOLISM, Issue 3 2006M. González-Ortiz Aim:, To identify the effect of a nutritional liquid supplement designed for the patient with diabetes mellitus (Glucerna SR) in single administration on the postprandial glucose state, insulin secretion and insulin sensitivity in healthy subjects. Methods:, A randomized, single-blind, cross-over, clinical trial was carried out in 14 young, healthy, non-obese, volunteers. A basal metabolic profile, which included glucose level, insulin, total cholesterol, high-density lipoprotein and low-density lipoprotein cholesterol, triglycerides, creatinine, and uric acid, was measured. Subjects received a single administration of 300 kcal, gauged with water at 350 ml, of each of the following (at least 3 days apart): glucose 75 g, polymeric supplement (Ensure high calcium) 315 ml or Glucerna SR 323 ml. At the beginning of each administration and 30, 60, 90 and 120 min later, glucose and insulin concentrations were measured. Areas under the curve of glucose and insulin were calculated. First-phase and total insulin secretions and insulin sensitivity were also estimated. Results:, Glucose level at 120 min was significantly lower after receiving Ensure high calcium or Glucerna SR. Administration of Glucerna SR resulted in a significant reduction in the areas under the curve of glucose and insulin, as well as in total insulin secretion with a tendency to be lower in their first phase. Insulin sensitivity was increased. Conclusions:, A single administration of Glucerna SR to healthy subjects decreased the postprandial glucose and insulin states, as well as the insulin secretion; insulin sensitivity increased. [source] Activation of dorsal horn microglia contributes to diabetes-induced tactile allodynia via extracellular signal-regulated protein kinase signalingGLIA, Issue 4 2008Makoto Tsuda Abstract Painful neuropathy is one of the most common complications of diabetes, one hallmark of which is tactile allodynia (pain hypersensitivity to innocuous stimulation). The underlying mechanisms of tactile allodynia are, however, poorly understood. Emerging evidence indicates that, following nerve injury, activated microglia in the spinal cord play a crucial role in tactile allodynia. However, it remains unknown whether spinal microglia are activated under diabetic conditions and whether they contribute to diabetes-induced tactile allodynia. In the present study, using streptozotocin (STZ)-induced diabetic rats that displayed tactile allodynia, we found several morphological changes of activated microglia in the dorsal horn. These included increases in Iba1 and OX-42 labeling (markers of microglia), hypertrophic morphology, the thickness and the retraction of processes, and in the number of activated microglia cells. Furthermore, in the dorsal horn of STZ diabetic rats, extracellular signal-regulated protein kinase (ERK) and an upstream kinase, Src-family kinase (SFK), both of which are implicated in microglial functions, were activated exclusively in microglia. Moreover, inhibition of ERK phosphorylation in the dorsal horn by intrathecal administration of U0126, an inhibitor of ERK activation, produced a striking alleviation of existing, long-term tactile allodynia of diabetic rats. We also found that a single administration of U0126 reduced the expression of allodynia. Together, these results suggest that activated dorsal horn microglia may be a crucial component of diabetes-induced tactile allodynia, mediated, in part, by the ERK signaling pathway. Thus, inhibiting microglia activation in the dorsal horn may represent a therapeutic strategy for treating diabetic tactile allodynia. © 2008 Wiley-Liss, Inc. [source] Acute oral toxicity of colchicine in rats: effects of gender, vehicle matrix and pre-exposure to lipopolysaccharideJOURNAL OF APPLIED TOXICOLOGY, Issue 5 2007Paddy L. Wiesenfeld Abstract The oral toxicity of a single administration by gavage (10, 20 or 30 mg kg,1 body weight) of colchicine (COL) was determined in young, mature male and female Sprague-Dawley rats. The effect of COL was evaluated in the presence or absence of additional treatment variables that included vehicle and lipopolysaccharide (LPS) pre-exposure. The vehicle for COL was either Half and Half cream (H & H) or saline, and each group included pretreatment with either saline or a low, minimally toxic dose (83 µg kg,1 body weight) of LPS. Colchicine toxicity in both male and female age-matched rats was characterized by progressively more severe dose-related clinical signs of toxicity. These included mortality, decreased body weight and feed intake during the first several days after dosing, with recovery thereafter in surviving animals. There were differences in the severity of the toxic response to COL between male and female rats. The most notable sex-related difference was in COL lethality. Female rats were two times more susceptible to the lethal effects of COL than male rats. Saline or H & H delivery vehicles did not result in any apparent qualitative or quantitative differences in COL toxicity. LPS pretreatment significantly potentiated COL lethality in both males and females, although the potentiation in males was greater than in females. LPS pretreatment modestly increased the COL induced anorexic effect in surviving males, but not in surviving female animals. LPS did not appear to modulate either the body weights or clinical signs of COL induced toxicity in surviving males or females. Copyright © 2007 John Wiley & Sons, Ltd. [source] Intraperitoneal injection of d- galactosamine provides a potent cell proliferation stimulus for the detection of initiation activities of chemicals in rat liverJOURNAL OF APPLIED TOXICOLOGY, Issue 6 2005Yoshiji Asaoka Abstract In an in vivo 5-week initiation assay model, chemical hepatectomy by hepatotoxicant administration was utilized as a cell proliferation stimulus as an alternative to the two-thirds partial hepatectomy. The study investigated the effect of an intraperitoneal (i.p.) injection of d- galactosamine (d -gal) for this purpose in a medium-term liver bioassay, with a further focus on cell proliferation kinetics and cytochrome P450 (CYP) expression. In experiment I, cell proliferation in rat liver after a single administration of d -gal (700 mg kg,1, i.p.) was analysed by the bromodeoxyuridine (BrdU) labeling method, and CYP isozymes were quantified by immunoblotting. In experiment II, the induction of glutathione S-transferase placental form (GST-P) positive foci by 1,2-dimethylhydrazine (DMH) was evaluated in a modified in vivo 5-week initiation assay model. At 84 hours after single administration of d -gal (i.p.) the BrdU index was markedly elevated (27.5% ± 9.5%). Although CYP 2E1 and 1A2 apoprotein contents decreased transiently to less than 20% of the control level, subsequently they recovered to 60% and 40% of the control level, respectively, at 84 hours. Induction of GST-P positive foci in the group given DMH at 84 hours after a single administration of d -gal was significantly greater than in the control group, correlating with the kinetics of cell proliferation. In conclusion, the sensitivity of the present initiation assay using d -gal i.p. is high, so that d -gal i.p. can be considered an effective cell proliferation stimulus. Copyright © 2005 John Wiley & Sons, Ltd. [source] FGF-23 Is a Potent Regulator of Vitamin D Metabolism and Phosphate Homeostasis,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 3 2004Takashi Shimada Abstract We analyzed the effects of an FGF-23 injection in vivo. FGF-23 caused a reduction in serum 1,25-dihydroxyvitamin D by altering the expressions of key enzymes for the vitamin D metabolism followed by hypophosphatemia. This study indicates that FGF-23 is a potent regulator of the vitamin D and phosphate metabolism. Introduction: The pathophysiological contribution of FGF-23 in hypophosphatemic diseases was supported by animal studies in which the long-term administration of recombinant fibroblast growth factor-23 reproduced hypophosphatemic rickets with a low serum 1,25-dihydroxyvitamin D [1,25(OH)2D] level. However, there is no clear understanding of how FGF-23 causes these changes. Materials and Methods: To elucidate the molecular mechanisms of the FGF-23 function, we investigated the short-term effects of a single administration of recombinant FGF-23 in normal and parathyroidectmized animals. Results: An injection of recombinant FGF-23 caused a reduction in serum phosphate and 1,25(OH)2D levels. A decrease in serum phosphate was first observed 9 h after the injection and was accompanied with a reduction in renal mRNA and protein levels for the type IIa sodium-phosphate cotransporter (NaPi-2a). There was no increase in the parathyroid hormone (PTH) level throughout the experiment, and hypophosphatemia was reproduced by FGF-23 in parathyroidectomized rats. Before this hypophosphatemic effect, the serum 1,25(OH)2D level had already descended at 3 h and reached the nadir 9 h after the administration. FGF-23 reduced renal mRNA for 25-hydroxyvitamin D-1,-hydroxylase and increased that for 25-hydroxyvitamin D-24-hydroxylase starting at 1 h. In addition, an injection of calcitriol into normal mice increased the serum FGF-23 level within 4 h. Conclusions: FGF-23 regulates NaPi-2a independently of PTH and the serum 1,25(OH)2D level by controlling renal expressions of key enzymes of the vitamin D metabolism. In conclusion, FGF-23 is a potent regulator of phosphate and vitamin D homeostasis. [source] Amino Acid Absorption in Portal Blood After Duodenal Infusions of a Soy Protein Hydrolysate Prepared by a Novel Soybean Protease D3JOURNAL OF FOOD SCIENCE, Issue 7 2006Tomohiro Kodera ABSTRACT:, The intestinal absorption of amino acids from decapeptide was investigated in rats under unrestrained conditions. The soy protein hydrolysate utilized in the experiment was produced by a novel soybean protease D3. The enzymatic features of protease D3 showed high homology with cathepsin L and cathepsin K and the average molecular weight of D3 hydrolysate is approximately 1200. We compared the intestinal absorption of D3 hydrolysate in portal blood with that of an amino acids mixture and soy protein with the same amino acid composition by determining the concentration of individual amino acids after a single administration of a nitrogen source. The absorptive velocity and intensity of each amino acid were calculated from its rate of elevation in the portal blood. And in most cases, these were higher in the D3 hydrolysate than in amino acids mixture and protein. The proportion of the amount of each amino acid absorbed in portal blood from D3 hydrolysate was much more like the composition of the administrated amino acids than like that from the amino acids mixture. The result of in vitro digestion assay indicated that D3 hydrolysate was hydrolyzed easier than the hydrolysates produced by microbial proteases. This is the first report to demonstrate that the D3 hydrolysate, which contains decapeptide as a dominant fraction, was more rapidly utilized than the amino acids mixture and protein as is the case with di-, tripeptides. This suggested that this hydrolysate could be available for nutraceutical use as well as use in nutritious foods for athletes and patients. [source] New designer drug 1-(3,4-methylenedioxybenzyl) piperazine (MDBP): studies on its metabolism and toxicological detection in rat urine using gas chromatography/mass spectrometry,JOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 3 2004Roland F. Staack Abstract Studies are described on the metabolism and toxicological analysis of the piperazine-derived designer drug 1-(3,4-methylenedioxybenzyl)piperazine (MDBP) in rat urine using gas chromatography/mass spectrometry (GC/MS). The identified metabolites indicated that MDBP was metabolized by demethylenation and subsequent methylation to N -(4-hydroxy-3-methoxybenzyl)piperazine followed by partial glucuronidation or sulfation. Additionally, degradation of the piperazine moiety to N -(3,4-methylenedioxybenzyl)ethylenediamine and 3,4-methylenedioxybenzylamine and N -dealkylation to piperazine were observed. The authors' systematic toxicological analysis (STA) procedure using full-scan GC/MS after acid hydrolysis, liquid/liquid extraction and microwave-assisted acetylation allowed the detection of MDBP and its above-mentioned metabolites in rat urine after single administration of a dose calculated from the doses commonly taken by drug users. Assuming similar metabolism, the described STA procedure should be suitable for proof of an intake of MDBP by analysis of human urine. Copyright © 2004 John Wiley & Sons, Ltd. [source] Ovarian follicular development stimulated by leuprorelin acetate plus human menopausal gonadotropin in chimpanzeesJOURNAL OF MEDICAL PRIMATOLOGY, Issue 2 2005Nobuhiko Yoshimoto Abstract:, We attempted ovarian stimulation using gonadotropins in 14 chimpanzees. Subjects were given a single administration of leuprorelin acetate, followed by repeated administration of human menopausal gonadotropin (hMG) for 16,21 days. During the dosing period, the ovarian follicle diameter and count were measured by transvaginal ultrasonography. The hormone administration induced the development of multiple follicles, and multiple oocytes were subsequently retrieved. However, the follicle count was decreased, suggesting atresia, in some subjects. Statistically, the final follicle diameter was dependent on the dosing duration and the hMG dose in the late stage, while the maximum follicle count during hMG administration was dependent on age and the hMG dose in the early stage. Five subjects showed mild ovarian hyperstimulation syndrome (OHSS)-like symptoms with a high serum estradiol (E2) concentration. These results suggest that leuprorelin acetate plus hMG administration successfully stimulates the development of multiple ovarian follicles for oocyte retrieval and that the serum E2 concentration is predictive of OHSS-like symptoms in chimpanzees. [source] Increased methamphetamine-induced locomotor activity and behavioral sensitization in histamine-deficient miceJOURNAL OF NEUROCHEMISTRY, Issue 4 2002Yasuhiko Kubota Abstract We have recently suggested that the brain histamine has an inhibitory role on the behavioral effects of methamphetamine by pharmacological studies. In this study, we used the histidine decarboxylase gene knockout mice and measured the spontaneous locomotor activity, the changes of locomotion by single and repeated administrations of methamphetamine, and the contents of brain monoamines and amino acids at 1 h after a single administration of methamphetamine. In the histidine decarboxylase gene knockout mice, spontaneous locomotor activity during the dark period was significantly lower than in the wild-type mice. Interestingly, methamphetamine-induced locomotor hyperactivity and behavioral sensitization were facilitated more in the histidine decarboxylase gene knockout mice. In the neurochemical study, noradrenaline and O -phosphoserine were decreased in the midbrain of the saline-treated histidine decarboxylase gene knockout mice. On the other hand, single administration of methamphetamine decreased GABA content of the midbrain of the wild-type mice, but did not alter that of histidine decarboxylase gene knockout mice. These results suggest that the histamine neuron system plays a role as an awakening amine in concert with the noradrenaline neuron system, whereas it has an inhibitory role on the behavioral effects of methamphetamine through the interaction with the GABAergic neuron system. [source] Clinical application of an enzyme immunoassay for cholecystokinin-like immunoreactive substance for determination of the human plasma levels: the effect of metoclopramide on gastrointestinal peptides and stress-related hormonesJOURNAL OF PEPTIDE SCIENCE, Issue 5 2006Fumihiko Katagiri Abstract Metoclopramide, a prokinetic drug, is widely used to treat vomiting and nausea. Delayed gastric emptying and continual stress are considered important factors, among others, that induce nausea and vomiting. One gastrointestinal motility regulatory factor has been assumed to be the induction of changes in the levels of peptides such as gastrin, somatostatin, motilin, and cholecystokinin (CCK) in plasma. In contrast, adrenocorticotropic hormone (ACTH) and cortisol are used as indicators of stress. Here, we studied the effects of metoclopramide on human plasma gastrin-, somatostatin-, motilin-, and CCK-like immunoreactive substances (ISs) and ACTH-IS and cortisol under stress conditions using repetitive blood sampling in healthy subjects. Metoclopramide hydrochloride at a dose of 30 mg or placebo was orally administered to five healthy male volunteers. Blood samples were taken before and 20, 40, 60, 90, 120, 180, and 240 min after administration, subject to extracting procedures, and submitted to a highly sensitive enzyme immunoassay system. A single administration of metoclopramide caused significant increases in plasma somatostatin-IS levels compared with the placebo. Metoclopramide significantly decreased plasma gastrin- and suppressed ACTH-IS and cortisol levels compared with the placebo. We hypothesize that metoclopramide might have an accelerating gastric-emptying effect and a modulatory effect on the hypothalamo-pituitary-adrenal (HPA) axis and the autonomic nervous function. These effects might be beneficial in stress-related diseases, which suggest that this medicine has clinicopharmacological activities. Copyright © 2005 European Peptide Society and John Wiley & Sons, Ltd. [source] Repetitive administration of Shaoyao-Gancao-tang to rats restores the bioavailability of glycyrrhizin reduced by antibiotic treatmentJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 11 2003Ju-Xiu He ABSTRACT Shaoyao-Gancao-tang (SGT), a traditional Chinese formulation, is often used together with antibiotics such as amoxicillin and metronidazole (AMPC-MET) for the treatment of peptic ulcers in Japan. However, the bioavailability of glycyrrhizin (GL) in SGT is severely reduced by a single administration of AMPC-MET, and the reducing effect continues for 12 days. GL is one of the major pharmacologically important glycosides in SGT and is transformed into the active metabolite 18,-glycyrrhetic acid (GA) by intestinal bacteria in the gut, followed by absorption of the latter into the blood. In order to reduce the negative effect of AMPC-MET on the bioavailability of GL, the optimum scheduling of the medications was examined. We found that the reduction in the plasma GA concentration and the GL-metabolizing activity in faeces caused by a single dose of AMPC-MET could be sharply attenuated by the repetitive administration of SGT for 4 days. The GA concentration and the GL-metabolizing activity were strongly enhanced by further continuous administration of SGT. These findings suggest that repetitive administration of SGT starting 1 or 2 days after the administration of AMPC-MET speeds the recovery of the bioavailability of GL in SGT. Similar strategies for administering medications may also be useful for combination therapy of antibiotics with other traditional Chinese formulations containing bioactive glycosides. [source] A Post-ischaemic Single Administration of Galanthamine, a Cholinesterase Inhibitor, Improves Learning Ability in RatsJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 9 2000A. I. ILIEV Transient forebrain ischaemia is widely observed in clinical practice. We have examined the effect of a single administration of the Cholinesterase inhibitor galanthamine (2 mgkg,1, i.p.) 25 min after reperfusion in male Sprague-Dawley rats (180 ± 20 g) after a 20-min common carotid artery occlusion. Twenty-four-hours post-ischaemia there was no difference in motor co-ordination or muscle tonus of the rats treated with or without galanthamine as assessed by the rota-rod test. Learning ability was examined using the shuttle-box test, evaluating the latency time and the number of errors for six days in succession. The performance of the ischaemic saline-injected rats was significantly impaired on days 4, 5, 6 (latency time) compared with the non-ischaemic rats and with the ischaemic animals administered galanthamine (P< 0.05). Similar results were obtained when counting the number of errors (failure to cross the cage during conditioned or unconditioned stimulus). The monitoring of body temperature during the first 12-h post-ischaemia did not show any significant difference between the groups. The data showed a beneficial effect of galanthamine on the recovery of learning ability when administered once only post-ischaemia. This suggests a direct effect on the early pathologic mechanisms of CNS damage. Cholinesterase inhibitors may prove useful in the early clinical treatment of ischaemic conditions. [source] Enamel matrix derivative enhances tissue formation around scaffolds used for tissue engineering of ligamentsJOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE, Issue 2 2010Michael P. Messenger Abstract The following in vitro translational study investigated whether enamel matrix derivative (EMD), an approved biomimetic treatment for periodontal disease (Emdogain®) and hard-to-heal wounds (Xelma®), enhanced synovial cell colonization and protein synthesis around a scaffold used clinically for in situ tissue engineering of the torn anterior cruciate ligament (ACL). Synovial cells were enzymatically extracted from bovine synovium and dynamically seeded onto polyethylene terephthalate (PET) scaffolds. The cells were cultured in low-serum medium (0.5% FBS) for 4 weeks with either a single administration of EMD at the start of the 4 week period or multiple administrations of EMD at regular intervals throughout the 4 weeks. Samples were harvested and evaluated using the Hoechst DNA assay, BCA protein assay, cresolphthalein complexone calcium assay, SDS,PAGE, ELISA and electron microscopy. A significant increase in cell number (DNA) (p < 0.01), protein content (p < 0.01) and TGF,1 synthesis (p < 0.01) was observed with multiple administrations of EMD. Additionally, SDS,PAGE showed an increase in high molecular weight proteins, characteristic of the fibril-forming collagens. Electron microscopy supported these findings, showing that scaffolds treated with multiple administrations of EMD were heavily coated with cells and extracellular matrix (ECM) that enveloped the fibres. Multiple administrations of EMD to synovial cell-seeded scaffolds enhanced the formation of tissue in vitro. Additionally, it was shown that EMD enhanced TGF,1 synthesis of synovial cells, suggesting a potential mode of action for EMD's capacity to stimulate tissue regeneration. Copyright © 2009 John Wiley & Sons, Ltd. [source] Pharmacokinetic comparison of orally-disintegrating metoclopramide with conventional metoclopramide tablet formulation in healthy volunteersALIMENTARY PHARMACOLOGY & THERAPEUTICS, Issue 3 2009R. FASS Summary Background, Oral tablet formulations of metoclopramide are effective therapies for gastroparesis and gastro-oesophageal reflux disease; however, difficulty swallowing tablets or nausea/vomiting may reduce patient adherence to therapy. Because of this, a metoclopramide orally-disintegrating tablet (ODT) has been developed. Aim, To evaluate the bioequivalence of a single administration of a 10-mg metoclopramide ODT and a conventional 10-mg oral metoclopramide tablet in healthy volunteers. Methods, In a randomized, single-dose, crossover study, healthy volunteers received single administration of 10-mg metoclopramide ODT and 10-mg conventional metoclopramide tablet, with a 7-day interval between treatments. Serial blood samples were collected before dosing and during 24 h post-treatment. Results, Forty-one volunteers completed both treatment arms. Metoclopramide ODT was bioequivalent to conventional tablets; 90% CIs for geometric mean treatment ratios of Cmax [91.6% (90% CI, 87.7,95.8%)], AUClast [97.3% (90% CI, 94.5,100.2%)] and AUCinf [97.6% (90% CI, 94.5,100.8%)] were within the predefined range. Of the 44 volunteers included in the safety analysis, 9 (20%) reported AEs after ODT, compared with 13 (30%) after conventional tablets. Conclusion, In healthy volunteers, single administration of 10-mg metoclopramide ODT was well tolerated and bioequivalent to single administration of a conventional 10-mg metoclopramide tablet. [source] The pharmacokinetics and pharmacodynamics of alfaxalone in cats after single and multiple intravenous administration of Alfaxan® at clinical and supraclinical dosesJOURNAL OF VETERINARY PHARMACOLOGY & THERAPEUTICS, Issue 6 2008T. WHITTEM This study aimed to determine the pharmacokinetic parameters and pharmacodynamics of alfaxalone in a 2-hydroxypropyl-,-cyclodextrin alfaxalone formulation (Alfaxan®, Jurox Pty Ltd, Rutherford, NSW, Australia) in cats after single administration at clinical and supraclinical dose rates and as multiple maintenance doses. First, a prospective two-period cross-over study was conducted at single clinical and supraclinical doses. Second, a single group multiple dose study evaluated the effect of maintenance doses. Eight (five female and three male) domestic cats completed the cross-over experiment and six female cats completed the multiple dose study. In the first experiment, alfaxalone was administered intravenously (IV) at 5 or 25 mg/kg with a washout period of 14 days. In the second experiment, alfaxalone was administered IV at 5 mg/kg followed by four doses each of 2 mg/kg, administered at onset of responsiveness to a noxious stimulus. Blood was collected at prescribed intervals and analysed by LCMS for plasma alfaxalone concentration. Noncompartmental pharmacokinetics were used to analyse the plasma alfaxalone data. The plasma clearance of alfaxalone at 5 and 25 mg/kg differed statistically at 25.1 and 14.8 mL/kg/min respectively. The elimination half lives were 45.2 and 76.6 min respectively. Alfaxalone has nonlinear pharmacokinetics in the cat. Nevertheless, for cats dosed with sequential maintenance doses, a regression line through their peak plasma concentrations indicated that there was no clinically relevant pharmacokinetic accumulation. The duration of nonresponsiveness after each maintenance dose was similar at approximately 6 min, indicating a lack of accumulation of pharmacodynamic effect. The cardiovascular and respiratory parameters measured in cats after administration of the labelled doses of Alfaxan® were stable. In conclusion, the pharmacokinetics of alfaxalone in cats are nonlinear. At clinical dose rates, however, neither alfaxalone nor its effects accumulated to a clinically relevant extent. Further, in the un-premedicated cat the induction and maintenance of surgical anaesthesia was free of untoward events after a dose of 5 mg alfaxalone/kg body weight followed by four sequential doses of 2 mg/kg as needed (i.e., approximately 7 to 8 mg/kg/h). [source] Protease-activated receptor-4 (PAR4): a role as inhibitor of visceral pain and hypersensitivityNEUROGASTROENTEROLOGY & MOTILITY, Issue 11 2009C. Augé Abstract, Protease-activated receptor-4 (PAR4) belongs to the family of receptors activated by the proteolytic cleavage of their extracellular N-terminal domain and the subsequent binding of the newly released N-terminus. While largely expressed in the colon, the role of PAR4 in gut functions has not been defined. We have investigated the effects of PAR4 agonist on colonic sensations and sensory neuron signalling, and its role in visceral pain. We observed that a single administration of the PAR4 agonist peptide (AYPGKF-NH2), but not the control peptide (YAPGKF-NH2) into the colon lumen of mice significantly reduced the visceromotor response to colorectal distension at different pressures of distension. Further, intracolonic administration of the PAR4 agonist, but not the control peptide, was able to significantly inhibit PAR2 agonist- and transcient receptor potential vanilloid-4 (TRPV4) agonist-induced allodynia and hyperalgesia in response to colorectal distension. Protease-activated receptor-4 was detected in sensory neurons projecting from the colon, and isolated from the dorsal root ganglia, where it co-expressed with PAR2 and TRPV4. In total sensory neurons, PAR4 agonist exposure inhibited free intracellular calcium mobilization induced by the pro-nociceptive agonists of PAR2 and TRPV4. Finally, PAR4 -deficient mice experienced increased pain behaviour in response to intracolonic administration of mustard oil, compared with wild-type littermates. These results show that PAR4 agonists modulate colonic nociceptive response, inhibit colonic hypersensitivity and primary afferent responses to pro-nociceptive mediators. Endogenous activation of PAR4 also plays a major role in controlling visceral pain. These results identify PAR4 as a previously unknown modulator of visceral nociception. [source] Effects of Sairei-to on the pharmacokinetics of nifedipine in ratsPHYTOTHERAPY RESEARCH, Issue 1 2008Mika Ikehata Abstract Sairei-to is a traditional herbal medicine used to complement, and as an alternative to, Western drugs. The aim of this study was to evaluate the pharmacokinetic interactions between Sairei-to and nifedipine (NFP), a substrate for CYP3A, in rats. NFP-oxidizing activity and the pharmacokinetics of NFP were examined after a single or 1-week of administration of Sairei-to (EK-114). NFP-oxidizing activity was enhanced transiently around 24 h after a single administration of EK-114 (1400 mg/kg). In vivo, the first-pass metabolism of NFP increased in the small intestine at 24 h after the administration of EK-114, and this effect disappeared at 72 h. Co-administration of EK-114 tended to inhibit the metabolism of NFP. On the other hand, when EK-114 was given at a high dose (1400 mg/kg) for 1 week, the oxidation of NFP in the small intestine was inhibited, and Cmax and AUC after the oral administration of NFP increased. In addition, a clinical dose of EK-114 (140 mg/kg) did not alter the pharmacokinetics of NFP, regardless of the administration schedule. EK-114 was suggested to affect the metabolism of NFP. However, the CYP3A-mediated pharmacokinetic interaction on the concomitant use of EK-114 may not be clinically significant. Copyright © 2007 John Wiley & Sons, Ltd. [source] The anxiolytic effect of Sho-ju-sen, a Japanese herbal medicine, assessed by an elevated plus-maze test in micePHYTOTHERAPY RESEARCH, Issue 2 2001Hisashi Kuribara Abstract Sho-ju-sen (SK), a Japanese herbal medicine with a nourishing tonic action, is composed of a water extract of Kumazasa leaves (Sasa kurinensis Makino et Sibata) (SS), and ethanol extracts of Japanese red pine needles (Pinus densiflora Sieb. et Zucc) (PN) and Ginseng roots (Panax ginseng C. A. Meyer) (PX) in the ratio 8:1:1. In this study, an elevated plus-maze test in mice was carried out to assess whether SK had an anxiolytic effect. No significant change was observed in either the plus-maze or activity test following a single administration of SK (10 and 20,mL/kg p.o.). However, mice allowed a free intake of SK (10% solution) for 5 days and longer showed a significant prolongation of the time spent in the open arms (an anxiolytic effect), as long as that caused by the benzodiazepine anxiolytic diazepam (1,mg/kg p.o.). SK (1%, 3% and 30% solutions for 7 days) tended to develop the anxiolytic effect. Of the constituents of SK, SS (8% solution), but not PN (1% solution) or PX (1% solution), resulted in the anxiolytic effect. Except for a slight acceleration in the motor activity by PN (1% solution), no significant change in the motor activity was produced by any treatment with SK, SS or PX. The combined treatment of SK (10% solution) or SS (8% solution) with 1,mg/kg diazepam enhanced the anxiolytic effect. Flumazenil (0.1,mg/kg s.c.), a benzodiazepine receptor antagonist, alone did not change the time spent in the open arms. However, it completely reversed the anxiolytic effect of SK, SS and diazepam. The present results suggest that: (1) long-term treatment with SK develops an anxiolytic effect, (2) SS is the main constituent for the anxiolytic effect of SK, and (3) benzodiazepine receptors are involved in the anxiolytic effect of SK and SS. Copyright © 2001 John Wiley & Sons, Ltd. [source] Peripheral olfactory sensitivity in rodents after treatment with docetaxel,THE LARYNGOSCOPE, Issue 4 2010Frédéric Faure MD Abstract Objectives/Hypothesis: Clinical studies have documented that cytotoxic chemotherapy is often associated with body weight loss and decreased enjoyment of food. Besides taste, olfaction plays a role in food intake. We assessed whether systemic chemotherapeutic cancer treatment compromises olfactory function in rats and mice treated with docetaxel (Taxotere; Sanofi-Aventis, Paris, France). Study Design: Randomized, controlled trials on mice and rats. Methods: Male mice received a single and male rats either a single, two, or three docetaxel administrations. Olfactory function was tested by means of electroolfactograms (EOGs) from the chemosensory epithelium of the nasal septum and the endoturbinates. We evaluated and compared the magnitude of EOG responses evoked by different odorants recorded at different time points after treatment. Results: In both animal species, docetaxel administration reduced body weight gain, thus evidencing the general toxic effect of the drug. In both animal species, the olfactory mucosa remained responsive to stimulation of odorants during the whole course of experiment, but treatment revealed regional differences of docetaxel susceptibility and induced marked transitory electrophysiological changes. In mice and rats a significant transitory decrease in EOG response magnitude occurred after a single administration. Unexpectedly, in rats we also observed an increase of the olfactory response following the second administration of the drug. Conclusions: Docetaxel exerts a neurotoxic effect on olfactory epithelia of rodents at doses similar to human doses, thus inducing transitory functional alterations. Although moderate, they are consistent with the hypothesis of a dysfunction of olfactory function. Further experiments are needed to elucidate the origin of the electrophysiological effects and their impact on the olfactory perception. Laryngoscope, 2010 [source] Role of Cholecystokinin Receptors in Induction of Antinociception in Hot-Plate TestBASIC AND CLINICAL PHARMACOLOGY & TOXICOLOGY, Issue 2 2000Mehdi Rezayat In the present study, the antinociceptive effect of cholecystokinin receptor agonists in the hot-plate test in mice has been evaluated. Subcutaneous administration of cholecystokinin octapeptide (cholecystokinin-8; 0.001, 0.005, 0.01, 0.05, and 0.1 mg/kg), unsulfated cholecystokinin octapeptide (cholecystokinin-8U; 0.1 mg/kg) or caerulein (0.25 mg/kg) produced antinociception. Administration of the cholecystokinin tetrapeptide (cholecystokinin-4; 0.25, 0.5 and 1.0 mg/kg) had no effect in the hot-plate test. Subcutaneous injection of the selective cholecystokinin receptor antagonists, MK-329 (0.125, 0.25 and 0.5 mg/kg) or L-365,260 (0.125, 0.25 and 0.5 mg/kg), produced no antinociceptive response. When the animals were pretreated with the cholecystokinin receptor antagonists or naloxone (0.5 and 1 mg/kg), a significant decrease in the antinociceptive response induced by cholecystokinin-8 and caerulein was obtained. The results indicate that single administration of cholecystokinin receptor agonists could produce an antinociceptive effect which is probably mediated via cholecystokinin receptors. With respect to the results obtained from morphine and naloxone administration, it is concluded that there may be an interaction between cholecystokinin and opiate mechanisms. [source] Determination of curcumol in rat plasma by capillary gas chromatography with a hydrogen flame ionization detectorBIOMEDICAL CHROMATOGRAPHY, Issue 4 2010Xiao Zhao Abstract A simple and sensitive capillary gas chromatography with a hydrogen flame ionization detector (GC-FID) method was developed for the determination of curcumol in rat plasma. From a variety of compounds and solvents tested, buagafuran was selected as the internal standard (IS) and acetonitrile was found to be the best protein precipitation agent and solvent for extracting curcumol from plasma and tissues samples. (Buagafuran was used as an internal standard. Curcumol was extracted by a protein precipitation with acetonitrile.) The samples were determined by GC on an HP-5 column (30.0 m × 0.32 mm, 0.25 ,m); inlet volume 2 ,L; split ratio 10 : 1; inlet temperature 250°C; oven temperature 180°C; flow 1.0 mL/·min; FID 250°C; carrier gas N2. The resulting retention times of curcumol and IS were 6.0 and 9.5 min. There was good linearity over the range 0.133,133.3 ,g/mL (r = 0.9999) in plasma samples. The method recoveries were 97.7,102.0% in plasma, and the intra- and inter-day variances (RSD) were less than 15% in all cases. The GC method was applied to develop a pharmacokinetics study in which experimental rats received a single administration of curcumol by intravenous injection. Copyright © 2009 John Wiley & Sons, Ltd. [source] Determination of paclitaxel in human and rat blood samples after administration of low dose paclitaxel by HPLC-UV detectionBIOMEDICAL CHROMATOGRAPHY, Issue 3 2007Haruo Yonemoto Abstract A simple and sensitive HPLC-UV method was developed for the determination of paclitaxel (TXL) in human and rat blood samples. 4-Hydroxybenzoic acid n -hexyl ester was used as an internal standard. TXL was extracted by a liquid,liquid extraction with tert -butylmethyl ether. The disturbing peaks in the case of serum sample were removed by pre-extraction with hexane. The separation of TXL was achieved within 25 min using an ODS column with 50% acetonitrile aqueous solution as a mobile phase at a flow rate of 1.0 mL/min. The eluent was monitored at 230 nm, and the resulted retention times of TXL and IS were 11.2 and 20.4 min. The detection limits of TXL for human plasma, serum and rat plasma samples at a signal-to-noise ratio of 3 were 10, 9.5 and 7.5 ng/mL, respectively. The proposed methods were applicable to the determination of TXL in human patients' plasma ranging from 15 to 27 ng/mL. Furthermore, monitoring of the time course of TXL after its single administration to rat could be demonstrated. Copyright © 2007 John Wiley & Sons, Ltd. [source] A simple and sensitive HPLC-fluorescence method for quantification of MDMA and MDA in blood with 4-(4,5-diphenyl-1H -imidazol-2-yl)benzoyl chloride (DIB-Cl) as a labelBIOMEDICAL CHROMATOGRAPHY, Issue 12 2006Mamoru Tomita Abstract A sensitive high-performance liquid chromatographic method with fluorescence detection to determine 3,4-methylenedioxymethamphethamine (MDMA) and 3,4-methylenedioxyamphethamine (MDA) in human and rat whole blood or plasma samples was developed by using 4-(4,5-diphenyl-1H -imidazol-2-yl)benzoyl chloride (DIB-Cl) as a label. MDMA and MDA in a small amount of blood sample (ca 100 µL) were extracted by liquid,liquid extraction with ethyl acetate, and were derivatized with DIB-Cl under mild conditions (10 min at room temperature). A good separation of DIB-derivatives could be achieved within 45 min using a commercially available ODS column with an isocratic eluent of 10 mm citric acid,20 mm Na2HPO4 aqueous buffer (pH 4.0),CH3CN,CH3OH (50:45:5, v/v/v %). The calibration curves prepared with 1-methyl-3-phenylpropylamine (MPPA) as an internal standard showed good linearity (r = 0.999) with 0.36,0.83 ng/mL detection limit at a signal-to-noise ratio of 3. MDMA and MDA in rat whole blood could be monitored for 6 h after a single administration of MDMA (2.2 mg/kg, i.p.). The pharmacokinetic parameters for MDMA and MDA obtained by triplicate measurements were 426 ± 23 and 39 ± 6 ng/mL (Cmax), 20 ± 5 and 100 ± 10 min (Tmax), respectively. Copyright © 2006 John Wiley & Sons, Ltd. [source] Ibudilast in healthy volunteers: safety, tolerability and pharmacokinetics with single and multiple dosesBRITISH JOURNAL OF CLINICAL PHARMACOLOGY, Issue 6 2008Paul Rolan WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT , Ibudilast is an oral drug approved in Asia for asthma. , Tolerability of 10-mg regimens has been described previously. , Published pharmacokinetics (PK) are limited: single or 7-day repeat oral administration of 10 mg in healthy male Asian volunteers. WHAT THIS STUDY ADDS , Safety/tolerability and PK of a single 30-mg dose and a 30-mg twice daily (b.i.d.) 2-week regimen in male and female healthy volunteers. , Higher-dose regimens are relevant for testing in new neurological indications. , LC-MS/MS analytics for quantification of plasma and urine levels of ibudilast parent and its primary metabolite (6,7-dihydrodiol-ibudilast). AIMS To investigate the safety, tolerability and pharmacokinetics (PK) of ibudilast after a single-dose and a multiple-dose regimen. METHODS Healthy adult male (n = 9) and female (n = 9) volunteers were evaluated over a 17-day stay in a Phase 1 unit. Subjects were randomized 1 : 3 to either oral placebo or ibudilast at 30-mg single administration followed by 14 days of 30 mg b.i.d. Complete safety analyses were performed and, for PK, plasma and urine samples were analysed for ibudilast and its major metabolite. RESULTS Ibudilast was generally well tolerated. No serious adverse events occurred. Treatment-related adverse events included hyperhidrosis, headache and nausea. Two subjects discontinued after a few days at 30 mg b.i.d. because of vomiting. Although samples sizes were too small to rule out a sex difference, PK were similar in men and women. The mean half-life for ibudilast was 19 h and median Tmax was 4,6 h. Mean (SD) steady-state plasma Cmax and AUC0,24 were 60 (25) ng ml,1 and 1004 (303) ng h ml,1, respectively. Plasma levels of 6,7- dihydrodiol-ibudilast were approximately 30% of the parent. CONCLUSIONS Ibudilast is generally well tolerated in healthy adults when given as a single oral dose of 30 mg followed by 30 mg b.i.d. (60 mg day,1) for 14 days. Plasma PK reached steady state within 2 days of starting the b.i.d. regimen. Exposure to ibudilast was achieved of a magnitude comparable to that associated with efficacy in rat chronic pain models. [source] Pharmacokinetics and pharmacodynamics of TF-505, a novel nonsteroidal 5,-reductase inhibitor, in normal subjects treated with single or multiple dosesBRITISH JOURNAL OF CLINICAL PHARMACOLOGY, Issue 3 2002Tomoe Fujita Aims To assess the tolerability, pharmacokinetics and pharmacodynamics of a novel nonsteroidal,, and,, noncompetitive,, inhibitor,, of,, type,, I,, and,, type,, II,, 5,-reductases, (,)-(S)-4-[1-[4-[1-(4-isobutylphenyl) butoxy]benzoyl]indolizin-3-yl]butyric acid (TF-505), after single and multiple oral doses in healthy volunteers. Methods In the single-dose study, six young adult males in each dose group received 25 mg or 50 mg of TF-505, and six older males (, 40 years) in each dose group received 75 mg or 100 mg of TF-505. The subjects were given the drug in ascending dose and in the fasting state. Six subjects also received 50 mg of TF-505 after breakfast in a two-period crossover manner. In the multiple-dose study, six older males in each dose group received 12.5 mg or 25 mg TF-505 after breakfast daily for 7 days. Plasma concentrations of TF-505, dihydrotestosterone (DHT) and testosterone were measured. The pharmacokinetics of TF-505 were analysed by a compartment model with first-order absorption, first-order elimination and a lag time. Pharmacokinetic and pharmacodynamic relationships were evaluated by indirect response modelling with inhibition of input. Results Maximum plasma concentration (Cmax) and the area under the concentration,time curve (AUC) increased proportionately after the single dose up to 50 mg and with the multiple doses. Linearity was not detected between 75 and 100 mg of TF-505. Dose dependency was also noted for the effect of TF-505 on DHT concentrations following single doses up to 50 mg and multiple doses. Plasma DHT concentrations decreased maximally to 58.2, 49.5, 54.2 and 49.8% of basal values at 8,12 h after single administration of 25, 50, 75 and 100 mg TF-505, respectively, and to 60.5 and 49.4% at the 7th and 5th dose following multiple doses of 12.5 and 25 mg TF-505, respectively. The predicted effect curves matched the observed data when the indirect response model was applied to the time course of the suppressant effect of TF-505 on plasma DHT concentrations after both the single and multiple studies. Fifty percent inhibitory concentrations (IC50) of 0.82, 1.48, 1.31 and 0.88 µ g ml,1, zero-order rate constants for the onset of plasma DHT concentration changes (kin) of 17.8, 17.4, 17.0 and 10.7% h,1 and first-order rate constants,, for,, increase,, in,, plasma,, DHT,, concentrations,, to,, basal,, values,, (kout),, of,, 0.17,,, 0.16,,, 0.17,, and,, 0.10 h,1,, for,, the,, single,, study,, at,, doses,, of,, 25,,, 50,,, 75,, and 100 mg, respectively, were attained. In the multiple-dose study, IC50s were 1.74 and 1.49 µg ml,1 for the 12.5 and 25 mg doses, respectively. No serious adverse events related to TF-505 were observed. Conclusions TF-505 was well tolerated in healthy male volunteers. Accumulation of TF-505 in plasma was not observed during multiple dosing. The indirect response model described the relationships between pharmacokinetics and pharmacodynamics of TF-505. Such modelling is expected to yield an appropriate dosage regimen in subsequent clinical trials. [source] Novel function of DUSP14/MKP6 (dual specific phosphatase 14) as a nonspecific regulatory molecule for delayed-type hypersensitivityBRITISH JOURNAL OF DERMATOLOGY, Issue 5 2007Y. Nakano Summary Background, Nonspecific unresponsive states of delayed-type hypersensitivity (DTH) to unrelated antigens are induced in mice by a single administration of hapten. In these studies, we found a unique regulatory mechanism of contact hypersensitivity (CHS) mediated by nonspecific suppressor factor (NSF) induced by the intravenous injection of hapten-conjugated syngeneic spleen cells. NSF is a , 45-kDa protein released from the macrophage-like suppressor cells and binds selectively to dendritic cells (DCs). Moreover, NSF-treated DCs release a second , 20-kDa NSF (NSFint). Objectives, To try and identify NSF and characterize its function. Methods, The suppressor activity was evaluated by inhibition of the passive transfer of CHS by the effector cells sensitized with hapten and the antigen-presenting cell (APC) activity of hapten-primed draining lymph node cells (DLNCs) to induce CHS. NSF-containing supernatants obtained from the culture of spleen cells from mice that had been injected intravenously with oxazolone-conjugated syngeneic spleen cells 7 days before were prepared and purified with a Green A dye-affinity column, DEAE column and Sephacryl S-200 column. Then, samples of molecular mass of , 45 kDa were separated by native-PAGE (polyacrylamide gel electrophoresis) and nonreducing sodium dodecyl sulphate (SDS)-PAGE. After confirming the suppressor activity of proteins of , 45 kDa separated by native-PAGE, samples were separated by nonreducing SDS-PAGE, transferred onto polyvinylidene difluoride membranes and analysed using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Results, Proteins of , 45 kDa eluted from a Sephacryl S-200 column and the slice of native-PAGE gel exhibited the strong suppressor activity. Analyses using MALDI-TOF mass spectrometry and MASCOT algorithm of the protein bands around 45 kDa separated by nonreducing SDS-PAGE identified NSF as a 22·5-kDa protein, dual specific phosphatase 14/MAP-kinase phophatase-6 (DUSP14/MKP6), which functions as a negative regulator of the MAP-kinase signalling. Western blot analyses revealed that recombinant DUSP14 (rDUSP14) exists as the mixture of 22·5-kDa monomer and 45-kDa dimer under nonreducing conditions, and monomers under reducing conditions. Treatment with rDUSP14 at 4 °C for 2 h suppressed the ability of effector cells to transfer CHS dose dependently and the APC function of DLNCs to induce CHS. Epicutaneous application of rDUSP14 immediately after challenge inhibited the subsequent CHS expression. rDUSP14 was bound specifically by major histocompatibility complex class II (Ia)-positive spleen cells (presumably DCs). The suppressor activity of NSF was neutralized by anti-DUSP14 monoclonal antibody. Expression of DUSP14 mRNA in the spleen was upregulated parallel to the unresponsive state induced by hapten-conjugated cells. NSF, NSFint and rDUSP14 exhibited the phosphatase activity towards p -nitrophenyl phosphate in vitro as alkaline phosphatase. Conclusions, These studies indicate for the first time that NSF is a dimer of DUSP14 secreted by macrophage-like suppressor cells by stimulation with hapten-conjugated cells and exerts a regulatory function on CHS through DCs as a secreted phosphatase. [source] | ||||||||||||||||||||||||||||