Irsogladine maleate (2,4-diamino-6-[2,5-dichlorophenyl]-s-triazine maleate; IM), an anti-peptic ulcer drug, may have a protective effect on the gastrointestinal mucosa. This study investigated the effects of IM on spontaneous colitis in interleukin-10 gene-deficient (IL-10−/−) mice. Five-week-old IL-10−/− mice were fed a control diet or one containing 100 ppm of IM for 10 weeks. Colonic tissues were evaluated morphologically and histologically. J774A.1 murine monocyte/macrophage cells were incubated with IM after lipopolysaccharide stimulation. mRNA expression was assessed by quantitative polymerase chain reaction (PCR) and protein concentration by enzyme-linked immunosorbent assay (ELISA). Colonic length, weight, and histological scores clearly demonstrated that spontaneous colitis was prevented in IL-10−/− mice fed a diet containing IM compared with those fed control diet. Levels of tumor necrosis factor-alpha (TNF-α) (−2.5-fold), IL-1β (−5.4), interferon-gamma (IFN-γ) (−4.5), IL-17 (−113.0), IL-12p35 (−21.0), IL-12p40 (−3.4), and IL-23p19 (−4.2) mRNA expression were significantly decreased in the colonic tissues of IM-treated animals, suggesting that oral treatment with IM suppressed the T-helper (Th)1/Th17 immune response in the colonic mucosa. An in vitro study using monocyte/macrophage cells to clarify the pharmacological action of IM indicated that IL-12p40 and IL-23p19 mRNA expression levels were dose-dependently decreased by IM treatment. ELISA showed that IL-12p40 and IL-23 protein secretion were significantly decreased by IM in a dose-dependent manner. Oral treatment with IM prevented spontaneous colitis in IL-10−/− mice by suppressing the colonic mucosal Th1/Th17 immune response through inhibition of IL-12 and -23 production in monocyte/macrophage cells.
Selective serotonin reuptake inhibitors (SSRIs) are prescribed for the treatment of depression worldwide. SSRIs are suspected to increase the risk of suicidal ideation and behavior (suicidality) in children, adolescents, and young adults. We examined the association between SSRI therapy and suicidality by applying a logistic regression model to age-stratified data from the Food and Drug Administration (FDA) Adverse Event Reporting System database. We attempted to mitigate the effect of patient-related factors by data subsetting. We selected case reports for SSRIs as referred to in the World Health Organization Anatomical Therapeutic Chemical classification code N06AB. The association between SSRIs and “suicidal events” or “self-harm events” was calculated as a reporting odds ratio (ROR) and adjusted for covariates by logistic regression. For subjects <18 years old (y.o.) the adjusted RORs (95% confidence interval) of SSRI therapy with suicidal events were 9.58 (8.97–10.23) in the whole data analysis and 4.64 (4.15–5.19) in the subset analysis; those with self-harm events were 31.40 (27.71–35.58) and 16.31 (13.12–20.29), respectively. Although the adjusted RORs were lower in the subset analyses than in the whole data analyses, both analyses indicated associations between SSRI treatment and suicidal and self-harm events. In both analyses these associations were stronger in the <18 y.o. group than other age groups. Children and adolescents should be closely monitored for the occurrence of suicidality when they are prescribed SSRIs. In addition, we found that data subsetting might mitigate the effect of an intrinsic risk among patients taking the suspected drug.
Vitiligo is an inflammatory skin disorder in which activated T cells play an important role in its onset and progression. Epigallocatechin-3-gallate (EGCG), the major chemical constituent of green tea, exhibits remarkable anti-oxidative and anti-inflammatory properties. EGCG administration has been confirmed to decrease the risk of vitiligo; however, the underlying mechanism is undetermined. In this study, we proved that EGCG directly inhibited the kinase activity of Janus kinase 2 (JAK2). In primary cultured human melanocytes, EGCG pre-treatment attenuated interferon (IFN)-γ-induced phosphorylation of JAK2 and its downstream signal transducer and activator of transcription (STAT)1 and STAT3 in a dose-dependent manner. We further examined the chemoattractant expression in melanocytes and demonstrated that EGCG significantly inhibited IFN-γ-induced expression of intracellular adhesion molecule (ICAM)-1, CXCL10, and monocyte chemotactic protein (MCP)-1 in human melanocytes. In addition, EGCG reduced the protein levels of the corresponding receptors including CD11a, CXCR3, and CCR2 in human T lymphocytes. As a consequence, adhesion of human T cells to melanocytes induced by IFN-γ was effectively suppressed by EGCG. Taken together, our results provided new evidence for the effectiveness of EGCG in vitiligo treatment and supported JAK2 as a molecular target for vitiligo medicine development.
Trachelospermi caulis is used widely as an herbal medicine in oriental countries to attenuate fever and pain. We wished to reveal the novel function of this herb and its active component on barrier function in intestinal epithelial cells. Monolayers of intestinal epithelial cells (Caco-2) were used to evaluate the transepithelial electrical resistance (TEER) and quantity of permeated ovalbumin (OVA) as indices of barrier function. T. caulis increased TEER values on cell monolayers and decreased OVA permeation across cell monolayers. To ascertain the active component of T. caulis, the extract was isolated to five fractions, and the effect of each of these fractions on intestinal barrier function examined. Chloroform and ethyl acetate fractions showed increased TEER values and decreased OVA flux. Chloroform and ethyl acetate fractions contained mainly trachelogenin and its glycoside, tracheloside. Trachelogenin increased TEER values and decreased OVA flux by enhancing the tight-junction protein occludin (but not tracheloside) in Caco-2 monolayers. These findings demonstrated that trachelogenin, an active component of T. caulis, might help to attenuate food allergy or inflammatory bowel disease through inhibition of allergen permeation or enhancement of the intestinal barrier.
The application of rapid microbiological methods (RMM) to bacterial monitoring in pharmaceutical manufacturing processes is now a key topic, since timely microbiological data are critical for product release, continuous process improvement and quality control. An automated, highly sensitive detection system has been developed which can measure the amount of ATP in a sample in 2 h with one hundredfold more sensitive than the conventional ATP method. One of the major subjects for adoption and implementation of RMM is how to set the criterion value for practical microbial control. This value was conventionally been set by experimental rule and indicated as the number of colonies counted after incubation in a particular medium. We have adopted a new approach to set a criterion value which enables assessment in whether the status of the object is normal or not. By setting this criterion value, it is possible to conduct the microbiological control with the intended probability of false-positive and false-negative. In this approach the probability distribution model of the measurement value of each object in a normal status has been established by performing repetitive measurement of each object. We have suggested and verified the probability distribution form of the ATP measurement value using measurement data of the standard bacterial solution of Staphylococcus aureus. The theoretical value of the model was in good agreement with the actual measured value. The results suggest it is possible to set an applicable management criterion value using this model and to conduct new microbiological monitoring using RMM.
α5β1 Integrin, a fibronectin receptor, is becoming a pertinent therapeutic target and a promising prognostic biomarker for cancer patients. The aim of this study was to functionalize an α5β1-specific fibronectin–mimetic peptide sequence KSSPHSRN(SG)5RGDSP (called PR_b) as a positron emission tomography (PET) probe. PR_b was modified by addition of a β-alanine residue, conjugated with 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA), and radiolabeled with 18F based on the chelation of 18F-aluminum fluoride. A control probe was produced by glycine to alanine substitution in the RGD motif of PR_b. Cell binding and blocking assays, autoradiographic evaluation of tissue binding and blocking, dynamic PET scans, and a biodistribution study were conducted using cell lines and murine tumor models with determined expression levels of α5β1 and other related integrins. 18F-PR_b was produced with a labeling yield of 22.3±1.9% based on 18F-F−, a radiochemical purity of >99%, and a specific activity of 30–70 GBq/µmol; it exhibited α5β1-binding activity and specificity in vitro, ex vivo, and in vivo, and had a rapid blood clearance and a predominant renal excretion pathway. In vivo α5β1-positive tumors could be clearly visualized by 18F-PR_b PET imaging. Both imaging and biodistribution studies suggested higher uptake of 18F-PR_b in α5β1-positive tumors than in α5β1-negative tumors and higher α5β1-positive tumor uptake of 18F-PR_b than the control probe. In contrast, there was no significant difference seen in the contralateral muscle uptake. A PET radioprobe, 18F-PR_b, was developed de novo and potentially can be used for noninvasive detection of α5β1 expression in tumors.
The aim of this study was to investigate the effect of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), an active form of vitamin D, on the oral absorption and disposition of adefovir dipivoxil (P-glycoprotein (P-gp) substrate) and its major active metabolite, adefovir (multidrug resistance-associated protein 4 (Mrp4) substrate), in rats. The pharmacokinetics of intravenous adefovir and oral adefovir dipivoxil was evaluated in control and 1,25(OH)2D3-treated rats. The intestinal absorption of adefovir dipivoxil was investigated through an in situ closed loop study, and the tissue distribution of adefovir after oral administration of adefovir dipivoxil was evaluated in the two groups. There was no significant difference in pharmacokinetic parameters of intravenous adefovir between the two groups. Importantly, the total area under the plasma concentration–time curve from time zero to time infinity (AUC), peak plasma concentration (Cmax) and extent of absolute oral bioavailability (F) of adefovir after oral administration of adefovir dipivoxil were significantly higher in 1,25(OH)2D3-treated rats than in control rats. In the in situ closed loop study, there was no significant difference in the remaining fraction of adefovir dipivoxil in the duodenum, jejunum and ileum loops between the two groups. In the tissue distribution study after oral administration of adefovir dipivoxil, the tissue-to-plasma partition coefficients of adefovir in the liver, brain, kidney, and intestine were significantly lower in the 1,25(OH)2D3-treated rats than in control rats. The present study indicates that 1,25(OH)2D3 treatment can enhance the oral absorption of adefovir dipivoxil, likely via the induction of basolateral Mrp4 function in rat intestine. However, the impact of 1,25(OH)2D3 treatment on the pharmacokinetics of intravenous adefovir was limited. These results could lead to further studies in clinically significant P-gp and/or MRP4-mediated 1,25(OH)2D3–drug interactions.
Juniperus phoenicea is a tree of the Cupressaceae family that is popularly known in the south of Tunisia because of its wide application in herbal medicine, including the use of its leaves to treat many diseases such as diarrhea, rheumatism, and intestinal disorders. The aim of this study was to evaluate the ulceroprotective and antioxidant activity of essential oil extracted from the leaves of J. phoenicea (EOJp) against hydrogen chloride (HCl)/ethanol-induced ulcers in rats. The antiulcer activities of 50, 75 and 100 mg/kg body weight (b.w.) EOJp were investigated on 0.3 M HCl/ethanol-induced ulcers in rats. The essential oil yield was 0.69% with 48 compounds; α-pinene was the principal component (20.24%). In vivo pretreatment with EOJp given orally provided dose-dependent protection against HCl/ethanol-induced gastric ulcers in rats. Furthermore, pretreatment with EOJp significantly decreased malondialdehyde (MDA) content and increased the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). The activity of the antiulcerogenic EOJp could be from synergistic antioxidant and anti-secretory effects. Oral use of EOJp has excellent preventive effects on induced gastric ulcers comparable to those of the proton pump inhibitor (PPI) omeprazole.
Regulation of blood glucose levels as a therapeutic strategy for cerebral ischemia plays an important role in suppressing neuronal damage. In particular, suppression of post-ischemic glucose intolerance improves cerebral ischemia. We have reported that cerebral ischemia induces glucose intolerance and an increase in plasma insulin levels. However, the mechanism of insulin secretion after cerebral ischemia is unclear. Nerve growth factor (NGF), a member of the neurotrophin family, has high affinity for tropomyosin-related kinase A (TrkA). NGF/TrkA signaling is associated with neuronal survival, differentiation, and function. Recently, NGF/TrkA signaling has been reported to be associated with insulin synthesis and secretion. In the present study, we evaluated the insulin content and expression of NGF/TrkA by immunofluorescence and Western blotting after middle cerebral artery occlusion (MCAO) as a cerebral ischemia model. At 6, 12, and 24 h after MCAO, insulin contents were increased in MCAO mice. The expression of NGF was increased at 6, 12, and 24 h, whereas the expression of TrkA tended to decrease in pancreas after MCAO. These results suggest that NGF/TrkA signaling is an important factor in cerebral ischemia-induced insulin synthesis and secretion in the pancreas.
Pemetrexed, a chemotherapeutic drug, is highly active in non-small cell lung cancer and malignant pleural mesothelioma. Unfortunately, rashes are more commonly associated with pemetrexed than other chemotherapies, and it is recommended that patients receive corticosteroids (8 mg/d of dexamethasone) for 3 d, including the day of pemetrexed administration (day 1). However, the efficacy of corticosteroids in this context has not been fully verified. In this retrospective study, we evaluated the medical records of 78 patients who received pemetrexed between April 2009 and March 2014, to confirm whether supplementary corticosteroids prevented rash development. The incidence of rash was lower in the 47 patients who received supplementary corticosteroids (after day 1) compared with the incidence among the 31 patients who did not receive supplementary corticosteroids (19.1% vs. 38.7%). The average cutoff dosage of supplementary corticosteroids on day 2 and day 3 was 1.5 mg/d of dexamethasone, as calculated using the receiver operating characteristic curve, and the odds ratio was 0.33 (95% confidence interval: 0.12–0.94). Administration of ≥1.5 mg of corticosteroids on day 2 and day 3 significantly reduced the severity of the rash compared to no supplementary treatment (grades 2/3, 13.3% vs. 33.3%, p<0.05). However, increasing the dose of corticosteroids had no additional effect on rash development. These results suggest that ≥1.5 mg of supplementary dexamethasone on day 2 and day 3 (in addition to day 1) may be necessary for preventing pemetrexed-induced rash, but high doses of dexamethasone (e.g., 8 mg/d) are unnecessary.
The distribution pattern of perivascular nerves in some branches of rat mesenteric arteries was studied. Mesenteric arteries isolated from 8-week-old Wistar rats were divided into the 1st-, 2nd-, and 3rd-order branches. The distribution of perivascular nerves in each branch was immunohistochemically evaluated using antibodies against neuropeptide Y (NPY), tyrosine hydroxylase (TH), calcitonin gene-related peptide (CGRP), substance P (SP), and neuronal nitric oxide synthase (nNOS). The density of NPY-, TH-, CGRP-, and SP-like immunoreactive (LI) nerves in the 2nd and 3rd branches was significantly greater than that in the 1st branch, and a negative relationship was found between nerve density and arterial diameter, except for TH-LI nerves. The density of NPY- and TH-LI nerves in all branches, which was similar, was greater than that of CGRP- (except for NPY-LI nerves in the 1st branch), SP-, or nNOS-LI nerves. Double immunostaining revealed that TH-LI nerves made contact with nNOS-LI, CGRP-LI, and SP-LI nerves and that CGRP-LI nerves made contact with TH-, NPY-, or nNOS-LI nerves, while TH-LI and CGRP-LI nerves nearly merged with NPY-LI and SP-LI nerves, respectively. These results suggest that the each branch of mesenteric arteries is densely innervated by vasoconstrictor nerves containing NPY, TH, and vasodilator CGRP nerves. They also suggest that the intense density of perivascular nerves in the 2nd and 3rd branches may contribute to maintaining vascular tone.
We previously demonstrated that rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), protects against N-methyl-D-aspartic acid (NMDA)-induced retinal neurotoxicity, but the mechanism underlying this protection is not fully understood. The present study aimed to examine the effects of everolimus, another inhibitor of mTOR, on neuronal cell loss and inflammation in a rat model of NMDA-induced retinal neurotoxicity, and to determine whether the extracellular signal-regulated kinase (ERK) pathway contributes to the protective effect of everolimus. Intravitreal injection of NMDA (200 nmol) resulted in (1) cell loss in the ganglion cell layer, (2) increase in the numbers of CD45-positive leukocytes and Iba1-positive microglia, and (3) phosphorylation of ribosomal protein S6 (pS6), a downstream indicator of mTOR activity. Simultaneous injection of everolimus with NMDA significantly attenuated these NMDA-induced responses. The neuroprotective effect of everolimus was almost completely prevented by the mitogen-activated protein kinase/ERK kinase inhibitor U0126 (1 nmol). NMDA increased the level of phosphorylated ERK (pERK) in Müller cells, and increase in pERK levels was also observed after co-injection of NMDA and everolimus. These results suggest that everolimus has a neuroprotective effect against NMDA-induced retinal neurotoxicity, an effect that seems to be mediated partly by activation of the ERK pathway in Müller cells.
The root bark of Morus alba is commonly used as an alternative medicine due to its numerous health benefits in humans. However, the antidepressant effects of various active components from M. alba have not been fully elucidated. In this study, we aimed to determine whether sanggenon G, an active compound isolated from the root bark of M. alba, exhibited antidepressant-like activity in rats subjected to forced swim test (FST)-induced depression. Acute treatment of rats with sanggenon G (30 mg/kg, intraperitoneally (i.p.)) significantly reduced immobility time and increased swimming time without any significant change in climbing. Rats treated with sanggenon G also exhibited a decrease in the limbic hypothalamic-pituitary-adrenal (HPA) axis response to the FST, as indicated by attenuation of the corticosterone response and decreased c-Fos immunoreactivity in the hypothalamic paraventricular nucleus (PVN). In addition, the antidepressant-like effects of sanggenon G were significantly inhibited by WAY100635 (1 mg/kg, i.p.; a selective 5-hydroxytryptamine1A (5-HT1A) receptor antagonist), but not SCH23390 (0.05 mg/kg, i.p.; a dopamine D1 receptor antagonist). Our findings suggested that the antidepressant-like effects of sanggenon G were mediated by an interaction with the serotonergic system. Further studies are needed to evaluate the potential of sanggenon G as an alternative therapeutic approach for the treatment of depression.
Candida albicans is the most common cause of invasive fungal infections in humans. The C. albicans cell wall proteins play an important role in crucial host-fungus interactions and might be ideal vaccine targets to induce protective immune response in host. Meanwhile, protein that is specific to C. albicans is also an ideal target of vaccine. In this study, 11 proteins involving cell wall biosynthesis, yeast-to-hypha formation, or specific to C. albicans were chosen and were successfully cloned, purified and verified. The immune protection of vaccination with each recombinant protein respectively in preventing systemic candidiasis in BALB/c mice was assessed. The injection of rPmt4p vaccination significantly increased survival rate, decreased fungal burdens in the heart, liver, brain, and kidneys, and increased serum levels of both immunoglobulin G (IgG) and IgM against rPmt4p in the immunized mice. Histopathological assessment demonstrated that rPmt4p vaccination protected the tissue structure, and decreased the infiltration of inflammatory cells. Passive transfer of the rPmt4p immunized serum increased survival rate against murine systemic candidiasis and significantly reduced organ fungal burden. The immune serum enhanced mouse neutrophil killing activity by directly neutralizing rPmt4p effects in vitro. Levels of interleukin (IL)-4, IL-10, IL-12p70, IL-17A and tumor necrosis factor (TNF)-α in serum were higher in the immunized mice compared to those in the adjuvant control group. In conclusion, our results suggested that rPmt4p vaccination may be considered as a potential vaccine candidate against systemic candidiasis.
The multikinase inhibitor sorafenib has been used in the treatment of hepatocellular carcinoma, renal cell carcinoma, and differentiated thyroid carcinoma. Here we have demonstrated the production of the first specific antibody against sorafenib. Anti-sorafenib serum was obtained by immunizing mice with an antigen conjugated with bovine serum albumin and carboxylic modified 4-(4-aminophenoxy)-N-methyl-2-pyridinecarboxamide (AMPC) using the N-succinimidyl ester method. Enzyme labeling of sorafenib with horseradish peroxidase was similarly performed using carboxylic modified AMPC. A simple competitive enzyme-linked immunosorbent assay (ELISA) for sorafenib was developed using the principle of direct competition between sorafenib and the enzyme marker for anti-sorafenib antibody, which had been adsorbed by the plastic surface of a microtiter plate. Serum sorafenib concentrations lower than 0.04 µg/mL were reproducibly measurable using the ELISA. This ELISA was specific to sorafenib and showed very slight cross-reactivity (2.5%) with a major metabolite, sorafenib N-oxide. The values of serum sorafenib levels from 32 patients measured by this ELISA were comparable with those measured by HPLC, and there was a strong correlation between the values determined by the two methods (Y=1.016X−0.137, r=0.979). The specificity and sensitivity of the ELISA for sorafenib should provide a valuable new tool for use in therapeutic drug monitoring and pharmacokinetic studies of sorafenib.
We studied the effects of twelve 5,7-dihydroxyflavone analogs on adipogenesis in 3T3-L1 cells. Among the compounds, luteolin, diosmetin, and chrysoeriol partly inhibited adipogenesis by blocking the accumulation of triacylglycerol in the cells. Conversely, tricetin facilitated triacylglycerol accumulation in the cells. The induction of lipogenesis or lipolysis may depend on the number and bonding position of hydroxyl or methoxy groups on the B ring of 5,7-dihydroxyflavone. The mRNA expression levels of adipogenic and lipogenic genes were suppressed by luteolin treatment in the cells, while the mRNA levels of lipolytic genes were not affected. However, the expression levels of the adipogenic, lipogenic, and lipolytic genes, except for adipocyte protein 2 (aP2), were not affected by the addition of tricetin. Moreover, luteolin suppressed glucose transporter type 4 (GLUT4) gene and protein levels. These results indicate that luteolin decreased triacylglycerol levels in 3T3-L1 cells during adipogenesis through the suppression of adipogenic/lipogenic and GLUT4 genes and GLUT4 protein.
Atrial natriuretic peptide (ANP) plays an important role in vascular functions such as blood pressure regulation and relaxant activity. Individual vascular beds exhibit differences in vascular reactivity to various ligands, however, the difference in responsiveness to ANP between carotid and renal arteries and the molecular mechanisms of its vasorelaxant activity in a pathophysiological state, including hypertension, remain unclear. We therefore investigated this issue by exposing carotid and renal artery rings obtained from spontaneously hypertensive rats (SHR) to ANP. In the SHR artery (vs. control WKY artery), the ANP-induced relaxations were reduced in carotid artery but not renal artery. Acetylcholine-induced relaxations were reduced in both arteries in SHR (vs. WKY). Sodium nitroprusside-induced relaxation was similar in both arteries between the groups. In carotid arteries, the ANP-induced relaxation was not affected by endothelial denudation or by treatment with inhibitors of nitric oxide synthase, cyclooxygenase, the voltage-dependent potassium channel, or ATP-sensitive potassium channel in arteries from both SHR and WKY. In the carotid artery from WKY but not SHR, the ANP-induced relaxation was significantly reduced by inhibition of the large-conductance calcium-activated potassium channel (BKCa). The BKCa activator-induced relaxation was reduced in the SHR artery (vs. WKY). These results suggest that ANP-induced relaxation is impaired in the carotid artery from SHR and this impairment may be at least in part due to the reduction of BKCa activity rather than endothelial components.
Orthovanadate (OVA), a protein tyrosine phosphatase inhibitor, induces vasoconstriction in a Rho kinase-dependent manner. The aim of this study was to determine the mechanism underlying OVA-induced vasoconstriction of rat mesenteric arteries. OVA-induced constriction of mesenteric arterial rings treated with NG-nitro-L-arginine methyl ester (L-NAME, 0.1 mM), a nitric oxide synthase inhibitor, was significantly blocked by the Rho kinase inhibitor Y-27632 (R-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, 10 µM), extracellular signal-regulated kinase 1 and 2 (Erk1/2) inhibitor FR180204 (5-(2-phenyl-pyrazolo[1,5-a]pyridin-3-yl)-1H-pyrazolo[3,4-c]pyridazin-3-ylamine, 10 µM), Erk1/2 kinase (MEK) inhibitor PD98059 (2′-amino-3′-methoxyflavone, 10 µM), epidermal growth factor receptor (EGFR) inhibitor AG1478 (4-(3-chloroanilino)-6,7-dimethoxyquinazoline, 10 µM), and Src inhibitor PP2 (4-amino-3-(4-chlorophenyl)-1-(t-butyl)-1H-pyrazolo[3,4-d]pyrimidine, 3 µM). However, the myosin light chain kinase inhibitor ML-7 (1-(5-iodonaphthalene-1-sulfonyl)-homopiperazine, 10 µM) did not affect OVA-induced constriction. Phosphorylation of myosin phosphatase target subunit 1 (MYPT1, an index of Rho kinase activity) was abrogated by inhibitors of Src, EGFR MEK, Erk1/2, and Rho kinase. OVA-stimulated Erk1/2 phosphorylation was blocked by inhibitors of EGFR, Src, MEK, and Erk1/2 but not affected by an inhibitor of Rho kinase. OVA-induced Src phosphorylation was abrogated by an Src inhibitor but not affected by inhibitors of EGFR, MEK, Erk1/2, and Rho kinase. In addition, the metalloproteinase inhibitor TAPI-0 (N-(R)-[2-(hydroxyaminocarbonyl)methyl]-4-methylpentanoyl-L-naphthylalanyl-L-alanine amide, 10 µM) and an inhibitor of heparin/epidermal growth factor binding (CRM 197, 10 µg/mL) did not affect OVA-induced contraction of rat mesenteric arterial rings. These results suggest that OVA induces vasoconstriction in rat mesenteric arteries via Src, EGFR, MEK, and Erk1/2 activation, leading to the inactivation of myosin light chain phosphatase through phosphorylation of MYPT1.
Antibiotic concentrations must be maintained at an adequate level throughout cardiovascular surgery to prevent surgical site infection. This study aimed to determine the most appropriate timing for intraoperative repeated dosing of ampicillin–sulbactam, a commonly used antibiotic prophylaxis regimen, to maintain adequate concentrations throughout the course of cardiovascular surgery with cardiopulmonary bypass (CPB). The total plasma concentrations of ampicillin were monitored in 8 patients after ampicillin (1 g)–sulbactam (0.5 g) administration via initial intravenous infusion and subsequent CPB priming. Pharmacokinetic parameters were estimated and used to predict the free plasma concentrations of ampicillin. The mean values for the volume of distribution, elimination rate constant, elimination half-life, and total clearance of ampicillin were 15.8±4.1 L, 0.505±0.186 h−1, 1.52±0.47 h, and 7.72±2.72 L/h, respectively. When ampicillin (1 g)–sulbactam (0.5 g) was intravenously administered every 3, 4, 6, and 12 h after the start of CPB, the predicted free trough plasma concentrations of ampicillin were 15.20, 8.25, 2.74, and 0.13 µg/mL, respectively. Therefore, an every-6-h regimen was needed to maintain the free ampicillin concentration at more than 2 µg/mL during cardiovascular surgery with CPB. We suggest that the dose and dosing interval for ampicillin–sulbactam should be adjusted to optimize the efficacy and safety of treatment, according to the minimum inhibitory concentrations for methicillin-sensitive Staphylococcus aureus isolates at each institution. Registration number: UMIN000007356.
We describe a new method for affinity selection of peptide binders for soluble protein targets using magnetic beads via organic phase separation (MOPS) from a phage display library. As a model target molecule, a mouse monoclonal antibody against human integrin α9β1 (Y9A2) immobilized onto protein G magnetic beads was incubated with a 15-mer or 20-mer random peptide phage-display library. The suspensions containing the phage-magnetic beads conjugates were then transferred onto the organic phase and centrifuged in order to recover the Y9A2 bound phage immobilized on the protein G magnetic beads in the lower organic phase. After three rounds of biopanning, we were able to isolate specific phage clones that could not be obtained by the conventional approach. Furthermore, this new approach was found to be highly effective for isolating phage-binders for Fc-fusion constructs; indeed, enrichment of specific phage-binders was observed after only the first panning cycle. Thus, MOPS can improve the selection of specific phage-binders for soluble protein targets mainly due to the removal of non-specific binders.
Overuse of vitamin A as a dietary supplement is a concern in industrialized countries. High-level dietary vitamin A is thought to shift immunity to a T helper 2 (Th2)-dominant one, resulting in the promotion of allergies. We have been studying a fluorescein isothiocyanate (FITC)-induced contact hypersensitivity (CHS) mouse model that involves Th2-type immunity. We fed a diet with a high retinyl palmitate content (250 international units (IU)/g diet) or a control diet (4 IU/g diet) to BALB/c mice for three weeks. No augmentation of FITC-induced CHS was found in mice fed the diet with a high vitamin A content, although accumulation of the vitamin was confirmed in the livers of these animals. The results indicated that relatively short-term feeding of the high-level vitamin A diet did not influence the Th2-driven response at a stage with significant retinol accumulation in the liver. The results were in contrast to the high-dose pyridoxine diets that produced a reduced response in FITC-induced CHS.