Development and validation of a stability-indicating LC-UV and LC-MS/MS methods for quantitative analysis of anisomycin and identification of degradation products

ABSTRACT Multifunctional drug anisomycin was subjected to forced degradation in accordance with International Conference on Harmonisation (ICH) guidelines for the first time. The drug was exposed to the recommended stress conditions of hydrolysis (acidic, alkaline and neutral), oxidation, thermal stress and photolysis, in order to investigate its stability. Optimized LC-MS/MS method was validated as recommended by ICH Q2(R1) guideline with respect to the specificity, accuracy, precision, limits of detection and quantitation, linearity and robustness. Anisomycin exhibited high instability under alkaline and thermal (neutral hydrolysis) conditions. It showed moderate stability under acidic, neutral, oxidative, thermal (acidic hydrolysis) and photolytic conditions, with the lowest degradation level observed in the case of light and oxidation stress. Formation of the same degradation product, identified as deacetylanisomycin, was observed under all applied stress conditions.

Recombinant Human Thioredoxin-1 Protects Macrophages from Oxidized Low-Density Lipoprotein-Induced Foam Cell Formation and Cell Apoptosis

Oxidized low-density lipoprotein (ox-LDL)-induced macrophage foam cell formation and apoptosis play critical roles in the pathogenesis of atherosclerosis. Thioredoxin-1 (Trx) is an antioxidant that potently protects various cells from oxidative stress-induced cell death. However, the protective effect of Trx on ox-LDL-induced macrophage foam cell formation and apoptosis has not been studied. This study aims to investigate the effect of recombinant human Trx (rhTrx) on ox-LDL-stimulated RAW264.7 macrophages and elucidate the possible mechanisms. RhTrx significantly inhibited ox-LDL-induced cholesterol accumulation and apoptosis in RAW264.7 macrophages. RhTrx also suppressed the ox-LDL-induced overproduction of lectin-like oxidized LDL receptor (LOX-1), Bax and activated caspase-3, but it increased the expression of Bcl-2. In addition, rhTrx markedly inhibited the ox-LDL-induced production of intracellular reactive oxygen species (ROS) and phosphorylation of p38 mitogen-activated protein kinases (MAPK). Furthermore, anisomycin (a p38 MAPK activator) abolished the protective effect of rhTrx on ox-LDL-stimulated RAW264.7 cells, and SB203580 (a p38 MAPK inhibitor) exerted a similar effect as rhTrx. Collectively, these findings indicate that rhTrx suppresses ox-LDL-stimulated foam cell formation and macrophage apoptosis by inhibiting ROS generation, p38 MAPK activation and LOX-1 expression. Therefore, we propose that rhTrx has therapeutic potential in the prevention and treatment of atherosclerosis.

Consumption and expenditure on food prepared away from home among Mexican adults in 2006 / Consumo y gasto en alimentos preparados fuera de casa en adultos mexicanos en 2006

Objective. To describe food expenditure and consumption of foods prepared away from home among Mexican adults. Materials and methods. Data were from 45 241 adult participants in the National Health and Nutrition Survey 2006, a nationally-representative, cross-sectional survey of Mexican households. Descriptive statistics and multivariable linear and logistic regression were used to assess the relationship between location of residence, educational attainment, socioeconomic status and the following: 1) expenditure on all food and at restaurants, and 2) frequency of consumption of comida corrida or restaurant food and street food. Results. Food expenditure and consumption of food prepared away from home were positively associated with socioeconomic status, educational attainment, and urban vs. rural residence (p<0.001 for all relationships in bivariate analyses). Conclusions. Consumption of food prepared outside home may be an important part of the diet among urban Mexican adults and those with high socioeconomic status and educational attainment.

Objetivo. Describir los gastos en alimentos y el consumo de alimentos preparados fuera de casa en población mexicana. Material y métodos. Los datos fueron de 45 241 adultos mexicanos en la Encuesta Nacional de Salud y Nutrición de 2006, representativa al nivel nacional. Se utilizaron estadísticas descriptivas y regresión linear y logística para estimar la relación entre el lugar de residencia, el nivel educativo y el nivel socioeconómico, con el gasto en todos los alimentos y en restaurantes, y con la frecuencia de consumo de comida corrida, en restaurantes y de la calle. Resultados. El gasto en alimentos y el consumo de alimentos preparados se asociaron positivamente con el nivel socioeconómico, el nivel educativo y la residencia rural (p<0,001 para todas las relaciones). Conclusiones. El consumo de alimentos preparados puede ser una parte importante de la dieta de los adultos urbanos y de aquéllos con altos niveles socioeconómicos y educativos.

Expression of KATP in pulmonary artery smooth muscle cells under hypoxia-hypercapnia condition and the relationship with p38 MAPK pathway / 生理学报

The aim of the present study is to investigate the expressions of ATP-sensitive K(+) channels (KATP) in pulmonary artery smooth muscle cells (PASMCs) and the relationship with p38 MAPK signal pathway in rats. Male SD rat PASMCs were cultured in vitro, and a model of hypoxia and hypercapnia was reconstructed. PASMCs were divided to normal (N), hypoxia-hypercapnia (H), hypoxia-hypercapnia+DMSO incubation (HD), hypoxia-hypercapnia+SB203580 (inhibitor of p38 MAPK pathway) incubation (HS) and hypoxia-hypercapnia+Anisomycin (agonist of p38 MAPK pathway) incubation (HA) groups. Western blot was used to detect the protein expression of SUR2B and Kir6.1; semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the mRNA expression of SUR2B and Kir6.1. The results demonstrated that: (1) Compared with N, H, HD and HS groups, the expressions of Kir6.1 mRNA and protein in PASMCs of HA group were decreased significantly (P < 0.01), but there were no differences among N, H, HD and HS groups (P > 0.05); (2) Compared with N group, the expressions of SUR2B mRNA and protein in H, HD, HS and HA groups were increased significantly (P < 0.05), but there were no differences among H, HD, HS and HA groups (P > 0.05). The results imply that: (1) Hypoxia-hypercapnia, SB203580 didn't change the expressions of Kir6.1 mRNA and protein in PASMCs, but Anisomycin decreased the expressions of Kir6.1 mRNA and protein, so Kir6.1 may be regulated by the other subfamily of MAPK pathway; (2) Hypoxia-hypercapnia raised SUR2B mRNA and protein expressions in PASMCs, but SB203580 and Anisomycin did not affect the changes, so the increasing of SUR2B mRNA and protein induced by hypoxia-hypercapnia may be not depend on p38 MAPK pathway.

[ role of intrahippocampal microinjections of anisomycin on dexamethasone-induced modulation of memory consolidation in rats]

Evidence indicates that hippocampus and activation of glucocorticoid receptors in this area are necessary for emotional learning and memory processes; also some studies suggest that glucocorticoid's effects probably involve with processes of protein synthysis in the hippocmapus. The aim of this study was to determine the role of intrahippocampal microinjections of anisomycin [[AIMS] as a protein synthysis inhibitor]] on dexamethasone-induced modulation of memory consolidation in the passive avoidance learning [PAL] task in rats. In this study, 90 male Wistar rats [250-300 gr] were surgically implanted bilaterally with cannulae aimed at the dorsal hippocampus [DH] were trained in PAL task. In experiment 1. Dexamethasone [0.1, 0.5, 1 and 3 mg/kg IP] was injected immediately after training and vehicle injected into DH. In experiment 2. Anisomycin [0.5,1 micro g/ micro l/side] or vehicle were injected bilaterally into the DH followed immediately by IP injection of Dexamethasone [1 mg/kg] or vehicle. Two days after training, retention tests were done and step-through latency [STL] and total time spent in light chamber [TLC] of apparatus were recorded during 10 min and compared with controls. Data indicated that injection of Dexamethasone immediatly after training enhanced memory consolidation [P<0.01] and this effect was blocked by injection of ANS in to the DH [P<0.01]. The findings above showed that glucocorticoids play on important role in consolidation of emotional learning and probably in processes of protein synthesis in the hippocampus may play an important role in mediating these effects

Overactivated mitogen-activated protein kinase by anisomycin induces tau hyperphosphorylation / 生理学报

One of the pathological feathers of Alzheimer's disease (AD) is neurofibrillary tangles (NFTs), which consist of paired helical filaments (PHFs) formed by hyperphosphorylated microtubule-associated protein tau. To study the role of mitogen-activated protein kinase (MAPK) in tau hyperphosphorylation and the underlying mechanism, wild type mouse neuroblastoma cells (N2a) were dealt with different concentrations (0.1 microg/mL, 0.2 microg/mL and 0.4 microg/mL) of anisomycin (an activator of MAPK) for 6 h. The relationship between MAPK activity and tau phosphorylation at some Alzheimer-sites was analyzed, and the activities of protein kinase A (PKA) and glycogen synthase kinase-3 (GSK-3) were detected. The results showed that anisomycin activated MAPK in a dose-dependent manner, but tau hyperphosphorylation at Ser-198/199/202 and Ser-396/404 sites was only observed when the concentration of anisomycin was at the level of 0.4 microg/mL, and the alteration of tau phosphorylation at Ser-214 showed no significant difference in different groups. 0.2 microg/mL and 0.4 microg/mL of anisomycin led to an increase in the activity of GSK-3, respectively, but had no effect on the activity of PKA. Lithium chloride, a specific inhibitor of GSK-3, completely abolished the anisomycin-induced elevation of tau phosphorylation without any effect on the activity of MAPK. In conclusion, overactivation of MAPK up to a certain degree induces tau hyperphosphorylation at Ser-198/199/202 and Ser-396/404 sites, and this is probably related to the effect of activated GSK-3 by MAPK.

Role of c-Jun NH (2)-terminal kinase in insulin resistance after burn / 中华外科杂志

<p><b>OBJECTIVE</b>To investigate the role of c-Jun NH (2)-terminal kinase (JNk) in insulin resistance after burn and its mechanism.</p><p><b>METHODS</b>Twenty-four Sprague-Dawley rats were randomized to control, burn and burn + anisomycin groups. The rats in control group received sham burn trauma, and burn and burn + anisomycin groups received 30% total body surface area (TBSA) full thickness burn injury. Anisomycin (5 mg/kg) together with 250 microl dimethyl sulfoxide (DMSO) was injected to the rats in anisomycin group intravenously, and only 250 microl DMSO in the other two groups. Euglycemic-hyperinsulinemic glucose clamps was performed 2 hours after the injection. The changes of phospho-serine 307, phospho-tyrosine of insulin receptor substrate (IRS)-1 and phospho-JNK in muscle tissues were determined and compared using immunoprecipitation and Western blot analysis or immunohistochemistry in the three groups.</p><p><b>RESULTS</b>The infusing rates of total 10% glucose (mg x kg(-1) x min(-1)) in control, burn and burn + anisomycin group were 12.3 +/- 0.4, 6.6 +/- 0.3, 6.5 +/- 0.4, respectively. The level of IRS-1 Serine 307 phosphorylation and phospho-JNK in muscle increased significantly, while insulin-induced tyrosine phosphorylation of IRS-1 decreased markedly after burn.</p><p><b>CONCLUSIONS</b>The activation of JNK elevates the level of IRS-1 phospho-serine 307 and might play a role in insulin resistance after burn in rats.</p>

The translation inhibitor anisomycin induces Elk-1-mediated transcriptional activation of egr-1 through multiple mitogen-activated protein kinase pathways

The early growth response-1 gene (egr-1) encodes a zinc-finger transcription factor Egr-1 and is rapidly inducible by a variety of extracellular stimuli. Anisomycin (ANX), a protein synthesis inhibitor, stimulates mitogen-activated protein kinase (MAPK) pathways and thereby causes a rapid induction of immediate-early response genes. We found that anisomycin treatment of U87MG glioma cells resulted in a marked, time-dependent increase in levels of Egr-1 protein. The results of Northern blot analysis and reporter gene assay of egr-1 gene promoter (Pegr-1) activity indicate that the ANX- induced increase in Egr-1 occurs at the transcriptional level. Deletion of the serum response element (SRE) in the 5'-flanking region of egr-1 gene abolished ANX-induced Pegr-1 activity. ANX induced the phosphorylation of the ERK1/2, JNK, and p38 MAPKs in a time-dependent manner and also induced transactivation of Gal4-Elk-1, suggesting that Elk-1 is involved in SRE-mediated egr-1 transcription. Transient transfection of dominant-negative constructs of MAPK pathways blocked ANX-induced Pegr-1 activity. Furthermore, pretreatment with specific MAPK pathway inhibitors, including the MEK inhibitor U0126, the JNK inhibitor SP600125, and the p38 kinase inhibitor SB202190, completely inhibited ANX-inducible expression of Egr-1. Taken together, these results suggest that all three MAPK pathways play a crucial role in ANX-induced transcriptional activation of Pegr-1 through SRE-mediated transactivation of Elk

c-Jun N-terminal kinase is involved in motility of endothelial cell

Cell motility is essential for a wide range of cellular activities including anigogenesis as well as metastasis of tumor cells. Ras has been implicated in cell migration and invasion, and functions at upstream of mitogen-activated protein kinase (MAPK) families, which include extracellular-signal regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAPK. In the present study, we examined the role of JNK in endothelial cell motility using stable transfectant (DAR-ECV) of ECV304 endothelial cells expressing previously established oncogenic H-Ras (leu 61). DAR-ECV cells showed an enhanced angiogenic potential and motility (approximately 2-fold) compared to ECV304 cells. Western blot analysis revealed constitutive activation of JNK in DAR-ECV cells. Pretreatment of JNK specific inhibitors, curcumin and all trans-retinoic acid, decreased the basal motility of DAR-ECV cells in a dose-dependent manner. These inhibitors also suppressed the motility stimulated by known JNK agonists such as TNFalpha and anisomycin. To further confirm the role of JNK, ECV304 cells expressing dominant active SEK1 (DAS-ECV) were generated. Basal non-stimulated levels of the cellular migration were greater in DAS-ECV clones than those in control ECV304 cells. These results suggest that Ras-SEK1-JNK pathway regulates motility of endothelial cells during angiogenesis.

c-Jun N-terminal kinase is involved in motility of endothelial cell

Cell motility is essential for a wide range of cellular activities including anigogenesis as well as metastasis of tumor cells. Ras has been implicated in cell migration and invasion, and functions at upstream of mitogen-activated protein kinase (MAPK) families, which include extracellular-signal regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAPK. In the present study, we examined the role of JNK in endothelial cell motility using stable transfectant (DAR-ECV) of ECV304 endothelial cells expressing previously established oncogenic H-Ras (leu 61). DAR-ECV cells showed an enhanced angiogenic potential and motility (approximately 2-fold) compared to ECV304 cells. Western blot analysis revealed constitutive activation of JNK in DAR-ECV cells. Pretreatment of JNK specific inhibitors, curcumin and all trans-retinoic acid, decreased the basal motility of DAR-ECV cells in a dose-dependent manner. These inhibitors also suppressed the motility stimulated by known JNK agonists such as TNFalpha and anisomycin. To further confirm the role of JNK, ECV304 cells expressing dominant active SEK1 (DAS-ECV) were generated. Basal non-stimulated levels of the cellular migration were greater in DAS-ECV clones than those in control ECV304 cells. These results suggest that Ras-SEK1-JNK pathway regulates motility of endothelial cells during angiogenesis.