New, simple and specific spectrophotometric methods and artificial neural network (ANN) were developed and validated in accordance with ICH guidelines for the simultaneous estimation of Olmesartan (OLM), Amlodipine (AML), and Hydrochlorothiazide (HCT) in commercial tablets.-1 for AML, OLM and HCT, respectively. ANN  was also applied for the simultaneous determination of the three analytes in their combined pharmaceutical dosage form using spectral region from 230–340 nm.For spectrophotometric methods: First, Amlodipine (AML) was determined by direct spectrophotometry at 359 nm and by application of the ratio subtraction, the AML spectrum was removed from the mixture spectra. Then Hydrochlorothiazide (HCT) was determined directly at 315 nm without interference from Olmesartan medoxamil (OLM) which could be determined using the isoabsorptive method. The calibration curve is linear over the concentration range of 5–40, 2.5-40 and 2–40 μg mLThe proposed methods were successfully applied for the assay of the three analytes in laboratory prepared mixtures and combined pharmaceutical tablets with excellent recoveries. No interference was observed from common pharmaceutical additives. The results were favorably compared with those obtained by a reference spectrophotometric method. The methods are validated according to the ICH guidelines and accuracy, precision and repeatability are found to be within the acceptable limit. H-1,2,4-benzothiadiazine-7-sulphonamide-1,1-dioxide [Olmesratan medoxamil  model uv-1650 pc with quartz cell of 1 cm path length, connected to an IBM-compatible computer. The spectral bandwidth was 2 nm and wavelength-scanning speed 2800 nm/min. A uv lamp with a short wavelength (254 nm).t test, F test were performed using Microsoft® Excel. All calculations were performed using intel® core™ i5-2400, 3.10 GHz, 4.00 GB of RAM under Microsoft Windows 7.ANN was implemented in Matlab® 7.1.0.246 (R14) using Neural Network toolbox The OLM was obtained from AK Scientific Inc. . AML was obtained from Pfizer Inc. . HCT was kindly supplied by Al-Hekma pharmaceutical Company . The purities of OLM, AML and HCT were 99.5%, 99.5% and 99.78% respectively. Tribenzor® tablets 40/10/25  are label to contain 40 mg of OLM, 10 mg of AML base  and 25 mg of HCT (Batch number 134809). They were procured from U.S.A. market. Acetonitrile used throughout this study was of analytical grade.−1), AML (200 μg mL−1) and HCT (250 μg mL−1) were prepared by dissolving 12.5 mg of OLM, 10 mg of AML and 12.5 mg of HCT, separately in 50 mL acetonitrile. Stock solutions were stable for at least two weeks when stored refrigerated at 4°C.Stock solutions of OLM , AML (6.95 μg mL−1) and HTZ (12.5 μg mL−1). Spectral acquisition and the calculations were performed in the same manner as described in "Calibration procedures".Tribenzor® tablets were weighed and finely powdered. An accurately weighed portion of the powder equivalent to 40 mg of OLM, 13.9 mg of AML  and 25 mg of HCT was extracted into acetonitrile with the aid of sonication and the extract was filtered. The filtrate was diluted with acetonitrile to obtain final concentrations of 200, 69.5 and 125 μg mL-1 of AML and HCT, 2.5 - 40 μg mL -1 of OLM were transferred separately into a series of 5-mL volumetric flasks and the solutions were diluted to the volume with acetonitrile and mixed well. The zero order absorption spectra of the prepared solutions were recorded from 200 to 400 nm against acetonitrile as a blank and stored in the computer. Absorbance was measured at 359 nm, 315 nm and 260.5 nm (isoabsorpitive point) for determination of AML, HCT and total concentration of HCT and OLM respectively (OLM can be determined by subtraction). The calibration graphs were constructed by relating the absorbance at mentioned wavelengths to the corresponding concentrations of AML, HCT and OLM (or HCT) respectively. The regression equations for the data were computed.Aliquots of standard solutions equivalent to 5–40 μg mL Five level, three factor calibration design  was used“Calibration procedures -spectrophotometric methods” were applied. For the determination of HCT, the stored spectra of the laboratory prepared mixtures were divided by the spectrum of normalized AML (devisor), and the constant was subtracted from the ratio spectra followed by multiplication of the obtained spectra by the divisor. The result of these steps is the spectra of OLM and HCT binary mixture where HCT can be determined solely at 315 nm. Finally, absorbance was measured at 260 .5 nm  for determination of the total concentration of OLM and HCT (hence OLM by subtraction).Aliquots of AML, OLM and HCT are transferred from their standard working solution into a series of 5-mL measuring flasks, completed to volume with acetonitrile to prepare mixtures containing different ratios of AML, OLM and HCT. The spectra of these mixtures are scanned from 200–400 nm and stored in the computer. For the determination of AML, the same procedures under Artificial neural network (ANN) is a type of artificial intelligence method that resembles biological nervous system in having the ability to find the relationship between inputs and outputs. ANN is composed of elements called artificial neurons that are interconnected by connections called weights. Commonly neural networks are trained, so that a particular input leads to a specific target output. The network is adjusted, based on a comparison of the output and the target, until the network output matches the target. Typically many input/target pairs are used to train a network .ANN has a great advantage over other traditional multivariate methods in modeling linear and non-linear relationship between variables. There are many papers that describe the application of ANN on linear and non-linear data ,17. The •Optimization of parameters of ANNFor proper training of ANN model, several parameters have to be optimized. There are two transfer functions used in ANN, one between input and output of a node in the hidden layer and the other is applied in output layer. The use of these functions depends on relationship between the inputs and outputs. Tan sigmoid followed by purelin are commonly used for non-linear systems while purelin-purelin transfer functions are used for linear one (as in our case). Among other ANN parameters, the hidden neurons number (HNN) is related to the converging performance of the output error function during the learning process. The learning coefficient (Lc) controls the degree at which connection weights are modified during the learning phase. The learning coefficient decrease (Lcd) and learning coefficient increase (Lci) control the variation of Lc value. It varies as a function of performance of the ANN (the Lc decreases or increases with the mean square error). For optimization of the ANN parameters, many experiments have to be done through which we can improve the model performance. Optimized ANN parameters are summarized in Table Tribenzor® tablets are combined dosage form containing the calcium channel blocker AML, the angiotensin II receptor blocker OLM, and the diuretic HCT. It has been used in the treatment of hypertension. The ratio of AML: OLM: HCT in tablets is 1:4:2.5 respectively. This study was designed to develop simple, robust and accurate spectrophotometric and ANN methods for the simultaneous determination of three analytes in Tribenzor® tablets. Because of the practical simplicity, and wide availability of spectrophotometry in quality control laboratories, it was attempted in this study.0) absorption spectrum of AML, OLM and HCT (10 μg mL-1 for each) solutions are recorded against acetonitrile as a blank over the range of 200–400 nm. The absorption spectra of the three compounds, AML, OLM and HCT show highly overlapped spectral band in the region 200–300 nm  showed isoabsorptive point at 260.5 nm For AML: A315 = 0.010C2 + 0.001(r = 0.9999)For HCT: A260.5 = 0.041C3 + 0.077(r = 0.9989)For OLM: A1, C2 and C3 were the concentrations of AML, HCT and AML + OLM in μg mL-1, respectively, and r was the correlation coefficient. LOD and LOQ were calculated [where A was absorbance of the zero order absorption spectra, Clculated  accordinWhere, σ was the standard deviation of the intercept of regression line and S was the slope of regression line of the calibration curve. The results are given in Table The accuracy of the proposed spectrophotometric methods was tested by analyzing triplicate samples of standard AML, HCT and OLM solutions. The recovery percentages were 99.89 ± 0.937, 99.55 ± 1.889 and 101.96 ± 0.622% for AML, HCT and OLM, respectively  indicated the high accuracy and precision of the proposed methods, respectively  and relative standard deviations (RSD) proved the high specificity of the proposed methods Table .t − and F − values were less than the theoretical values [ANN, as a multivariate calibration method, is very useful in spectral analysis because the simultaneous inclusion of many spectral wavelengths instead of using a single wavelength greatly improves the precision and predictive ability of the method . The purl values  (Table 5From the previous discussion, it could be concluded that the proposed procedures are simple, rapid over chromatographic methods and it does not need sample preparation or sophisticated techniques and instruments. From the economic point of view, the analytical reagents are inexpensive and available in all laboratories. The validation of the proposed methods according to the ICH guidelines proved the applicability and great value of these methods for routine application in quality control laboratories for the simultaneous analysis of AML, OLM and HCT in their pure powder and dosage forms without prior separation or excipient interference.The author declares that he has no competing interest.


Large-scale pharmaco-epidemiological studies of Chinese herbal medicine (CHM) for treatment of urticaria are few, even though clinical trials showed some CHM are effective. The purpose of this study was to explore the frequencies and patterns of CHM prescriptions for urticaria by analysing the population-based CHM database in Taiwan.This study was linked to and processed through the complete traditional CHM database of the National Health Insurance Research Database in Taiwan during 2009. We calculated the frequencies and patterns of CHM prescriptions used for treatment of urticaria, of which the diagnosis was defined as the single ICD-9 Code of 708. Frequent itemset mining, as applied to data mining, was used to analyse co-prescription of CHM for patients with urticaria.Dictamnus dasycarpus Turcz) was the most commonly prescribed single Chinese herb while Xiao-Feng San was the most commonly prescribed Chinese herbal formula. The most commonly prescribed CHM drug combination was Xiao-Feng San plus Bai-Xian-Pi while the most commonly prescribed triple drug combination was Xiao-Feng San, Bai-Xian-Pi, and Di-Fu Zi (Kochia scoparia).There were 37,386 subjects who visited traditional Chinese Medicine clinics for urticaria in Taiwan during 2009 and received a total of 95,765 CHM prescriptions. Subjects between 18 and 35 years of age comprised the largest number of those treated (32.76%). In addition, women used CHM for urticaria more frequently than men . There was an average of 5.54 items prescribed in the form of either individual Chinese herbs or a formula in a single CHM prescription for urticaria. Bai-Xian-Pi (In view of the popularity of CHM such as Xiao-Feng San prescribed for the wind-heat pattern of urticaria in this study, a large-scale, randomized clinical trial is warranted to research their efficacy and safety. Urticaria is defined as a kind of skin rash notable for pale red, itchy bumps caused by allergic reactions to internal and external agents. The word ‘urticaria’ is derived from the Latin word urtica, which means ‘nettle’, which is a tooth-leaved plant covered with hairs that secret a stinging fluid that immediately affects the skin . AccordiUrticaria is a disorder affecting up to 25% of people in the United States (US). Chronic urticaria is defined as repeated episodes of symptoms that last for more than 6 weeks. The majority of cases (> 80%) have an unknown cause, which is called chronic idiopathic urticaria (CIU). The incidence of CIU is higher in women than in men (2:1), but not among atopic patients, with an evaluated prevalence of up to 1% in the US. CIU patients report quality of life impairment similar to patients who have cardiac disease and other chronic skin diseases such as atopic dermatitis and psoriasis. In addition, these patients who did not use immunosuppressants and who attended a university clinic had annual health care costs of more than US $2,000 .Urticaria is caused by an allergic response to an allergenic substance. Drugs commonly used for treating urticaria include antihistamines, omalizumab, cyclosporine, and low-dose corticosteroids . Side efThroughout the world, many people use complementary and alternative medicine, and Traditional Chinese Medicine (TCM) is one of the most popular forms. In Western populations, the reasons for the use of TCM include experiencing failure of standard health care, the need for autonomy, and preference for natural therapies -8. BasedThe National Health Insurance (NHI) program was initiated in Taiwan in 1995, and TCM has been covered by the NHI since 1996. Currently, the NHI covers about 99% of the 23 million population of Taiwan . CitizenBecause TCM is inexpensive and widely available, it has been used for the treatment of skin diseases for centuries. Additionally, some controlled clinical studies showed that TCM was effective and safe for treatment of inflammatory skin disorders . NonetheInternational Classification of Diseases, Ninth Revision, along with the prescription drugs and doses, special treatments, and dates of these orders.Taiwan began the National Health Insurance program in 1995 . The NatIn Taiwan, all TCMs are provided only in ambulatory clinics. In addition, only licensed TCM doctors are qualified for reimbursement . In thisIn Taiwan, TCM doctors are asked to make diagnoses based on ICD-9-CM coding. In this study, we used data from patients with the single diagnostic code for urticaria . From all 38,547,753 visits in the CM_CD2009.dat database, 37,386 subjects visited TCM clinics for urticaria in Taiwan during 2009. Among these subjects, a total of 95,765 prescriptions with CHM were recorded. The drug list for CHM was obtained from the Bureau of Health Promotion, and the database (CM_CD2009 and CM_OO2009) was interlinked with six variables  in the database.According to the TCM theory, a single prescription from a TCM doctor may contain four ingredients: 1) a single Chinese herb, 2) Fu-Fang, 3) Fang-Ji (regimen or remedy) or 4) Chia-Chien-Fang. Fu-Fang is composed of multiple herbs of various dosages. Fang-Ji is a combination of compatible Chinese herbs in fixed dosages according to classical or well-known Chinese textbooks of medicine. Chia-Chien-Fang is a classical formula in which a drug ingredient is combined with Chinese herbs. These ingredients are made of powder and can be easily mixed in a single prescription .Mining frequent itemsets and rules of association are popular and well researched methods for discovering interesting relations between variables in large databases. Piatetsky-Shaprio 1991) first described strong rules for database mining and analysis using measures of interestingness and association .These it91 first In this study, a data mining process was used to help discover and characterise prescription patterns of the Chinese herbal drugs or formulae for urticaria. The frequent itemset mining method was applied to evaluate co-prescriptions of CHM. The support (%) of a prescribed drug set X was defined as the proportion of all prescriptions in the data set that contained the drug set X. Support is a measure of how frequently the rule occurs in the database. The Statistical Software R (version 2.13.2) package ‘arules’ was used, and the function ‘apriori’ within a minimum support of 1% was set to perform the analyses.There were 37,386 subjects who visited TCM clinics for urticaria in Taiwan during 2009. Among these subjects, a total of 95,765 prescriptions for CHM were recorded. Almost two thirds of the patients treated for urticaria with CHM were between 18 and 50 years of age (Table Xiao-Feng San (48.84%) was by far the most commonly prescribed Chinese herbal formula for subjects with urticaria. The remaining half of prescriptions comprised 10 other CHM formulations Table .Dictamnus dasycarpus Turcz (Bai-Xian-Pi) (15.55%), Paeonia suffruticosa Andr (Mu-Dan-Pi) (13.21%), Kochia scoparia (Di-Fu-Zi) (12.75%) and Forsythia suspensa (Lian-Qiao) (12.50%). The prescription rates for each of the remaining herbs were less than 10% (Table The top four most commonly prescribed herbs for urticaria were Dan-Pi) 1.21%, KocDictamnus dasycarpus Turcz  IgE [Glycyrrhiza uralensis had macrophage immunomodulatory activity [In this study, Xiao-Feng San (48.84%) was by far the most commonly prescribed Chinese herbal formula for subjects with urticaria. According to the principles of TCM treatment for urticaria, Xiao-Feng San, which expels wind and clears heat, is prescribed for TCM patterns of Wind and Heat. Xiao-Feng San is famous for its antipruritic effect and is frequently used to treat chronic skin diseases such as urticaria . In a ra effects . An aqueDNP) IgE . Anotheractivity . On the activity .Dictamnus dasycarpus Turcz) (15.55%) was the most commonly used to treat urticaria. It is reported to have anti-allergic effects and directly inhibits scratching behaviour and vascular permeability induced by histamine and serotonin release [Paeonia suffruticosa Andr) (13.21%). According to TCM theory, Mu Dan Pi clears heat from the blood [P. suffruticosa Andr contains five compounds including paeonol, paeoniflorin, paeonoside, paeonollide and apiopaeonoside; among them, paeonol is the main bioactive component [With regard to a single Chinese herb, Bai-Xian-Pi ( release . The seche blood . P. suffomponent . It had omponent .Kochia scoparia) (12.75%) is the third commonly used Chinese herb in treating urticaria. The fruits of Kochia scoparia have been used in TCM for centuries to treat skin diseases. Momordin Ic is the main active component in the fruits of K. scoparia. Studies indicate that it has a peripheral antinociceptive effect mediated by anti-inflammatory action [K. scoparia is a potent inhibitor of lipopolysaccharide-induced nitric oxide, prostaglandin E2 and tumour necrosis factor alpha production [Forsythiae fructus, inhibits the effects of lipopolysaccharide by repressing a key inflammatory pathway related to NF-kB, prostaglandin E2 and nitric oxide production, and expression of proinflammatory cytokines [Our results showed that Di-Fu-Zi , inducible nitric oxide synthase (iNOS), and cytokines such as TNF-α, interleukin-1β and interleukin-6 by inhibiting the p38 mitogen-activated protein kinases and NF-κB pathways [[Other commonly prescribed Chinese herbs used for treatment of urticaria identified in our study included operties ,37. BesiIn this study, we explored the frequencies and patterns of CHM prescriptions for urticaria by analysing the population-based CHM database in Taiwan during 2009. Various CHM prescriptions are used to treat urticaria according to physicians’ personal experiences or based on traditional Chinese texts. However, it remains unclear which of the CHM prescriptions are the most effective in treating urticaria in clinical practice. We observed drug utilization and prescription patterns through a large scale survey of clinical practices, which served as an effective tool for investigating the clinical pharmacology of these compounds. In addition, this study provides relevant information to discern potentially effective CHM for treating urticaria. According to the principles of TCM diagnosis, syndrome differentiation and treatment, we could also summarize TCM patterns and explore the core patterns of urticaria from the results of this study. For example, if the symptoms include red wheals, aversion to wind, thirst, restlessness, a red tongue and rapid pulse, they would be subclassified as the Wind Heat pattern for which a formula such as Xiao-Feng San could be prescribed. On the other hand, if the symptoms include dry skin, pale face and lips, dizziness, a pale tongue and thin pulse, they would be subclassified as the Blood Deficiency pattern. Therefore, a formula such as Dang Gui Yin Zi would be administered. On the basis of the results of this study, future studies could concentrate on the most common TCM patterns found for urticaria, and different treatments could be designated for specific TCM patterns.Our study has two limitations. First, syndrome differentiation (bianzheng lunzhi) in TCM is the comprehensive analysis of clinical information gained by the four main diagnostic methods: inspection, auscultation or olfaction, inquiry, and palpation. They were written in the medical records by the treating physicians. The exact syndrome differentiation helps physicians select the right herbal formula to provide the best treatment for patients in certain phases of a disease. However, the identities of the patients were encrypted in the NHI reimbursement database; we could not obtain the medical records of the patients. Thus, we could not analyse the exact syndrome differentiation. Second, TCM patients might have received prior Western medicine treatment or be under concomitant therapies, and the therapeutic effect of Chinese medicine could be overestimated.In conclusion, we investigated health care claims data in a population-based pharmaco-epidemiology study of Chinese herbs for treating urticaria. The most commonly prescribed Chinese herbal formula for the treatment of urticaria is Xiao-Feng San, and the top three individual herbs are Bai-Xian-Pi, Mu Dan Pi and Di Fu Zi. The therapeutic effects and safety of these commonly used Chinese herbal formulae or individual herbs require further examinations through clinical studies or well-designed randomized, double blind, placebo-controlled trials.The authors declare that they have no competing interests.All authors were involved in the design and writing of the study, and SFW conducted the statistical analysis. All authors approved the submitted version of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/13/209/prepub


Here we tested the hypothesis that chronic fructose intake decreases 1,25(OH)2D3 levels independent of increases in Ca2+ requirements. Adult mice fed for five wk a high glucose-low Ca2+ diet displayed expected compensatory increases in intestinal and renal Ca2+ transporter expression and activity, in renal CYP27B1 (coding for 1α-hydroxylase) expression as well as in serum 1,25(OH)2D3 levels, compared with mice fed isocaloric glucose- or fructose-normal Ca2+ diets. Replacing glucose with fructose prevented these increases in Ca2+ transporter, CYP27B1, and 1,25(OH)2D3 levels induced by a low Ca2+ diet. In adult mice fed for three mo a normal Ca2+ diet, renal expression of CYP27B1 and of CYP24A1 (24-hydroxylase) decreased and increased, respectively, when the carbohydrate source was fructose instead of glucose or starch. Intestinal and renal Ca2+ transporter activity and expression did not vary with dietary carbohydrate. To determine the time course of fructose effects, a high fructose or glucose diet with normal Ca2+ levels was fed to adult rats for three mo. Serum levels of 1,25(OH)2D3 decreased and of FGF23 increased significantly over time. Renal expression of CYP27B1 and serum levels of 1,25(OH)2D3 still decreased in fructose- compared to those in glucose-fed rats after three mo. Serum parathyroid hormone, Ca2+ and phosphate levels were normal and independent of dietary sugar as well as time of feeding. Thus, chronically high fructose intakes can decrease serum levels of 1,25(OH)2D3 in adult rodents experiencing no Ca2+ stress and fed sufficient levels of dietary Ca2+. This finding is highly significant because fructose constitutes a substantial portion of the average diet of Americans already deficient in vitamin D.Excessive fructose consumption inhibits adaptive increases in intestinal Ca Fructose is one of the key components of the American diet and represents more than 10% of daily calorie intake 2+ levels in the serum due to low dietary intake or increased demand of Ca2+, the synthesis of the hormonally active form of vitamin D, 1,25-dihydroxyvitamin D3 2D3) is enhanced, leading to adaptive increases in intestinal Ca2+ absorption. If normal serum Ca2+ level cannot be maintained by intestinal absorption, then 1,25(OH)2D3 together with the parathyroid hormone (PTH) will mobilize bone Ca2+ and increase reabsorption of Ca2+ from the renal distal tubule 2+ transport involves Ca2+ entry through the apical Ca2+ channel transient receptor potential vanilloid 6 and 5 (TRPV6 and TRPV5) respectively, its intracellular diffusion via Ca2+-binding proteins (CaBP9k and CaBP28k respectively), and its extrusion across the basolateral membrane mainly through the plasma membrane Ca2+-ATPase (PMCA1). 1,25(OH)2D3 stimulates the transcription, via the vitamin D receptor (VDR) In the event of decreased Ca2D3 and Ca2+ metabolism 2+ transport, mainly by preventing the upregulation of TRPV6 and CaBP9k expression in the duodenum, and to a lesser extent, of TRPV5 and CaBP28k in the kidney. The mechanism underlying its inhibitory effect on Ca2+ transport is that chronic fructose intake also inhibits the adaptive increases in synthesis and blood levels of 1,25(OH)2D3 regulating intestinal and renal Ca2+ transport systems. Highlighting the key role of 1,25(OH)2D3, treatments with 1,25(OH)2D3 rescued the inhibitory effect of chronic fructose intake on intestinal Ca2+ absorption in weanling rats Our recent work has demonstrated that excessive fructose consumption affects 1,25(OH)3 (originating from the skin or diet) is initially hydroxylated by 25-hydroxylase (encoded by CYP2R1) in the liver, producing calcidiol or 25(OH)D3 that is further hydroxylated by 1α-hydroxylase (encoded by CYP27B1) to 1,25(OH)2D3 in the kidney. The circulating level of 1,25(OH)2D3 is balanced between synthesis by 1α-hydroxylase and degradation by 24-hydroxylase (encoded by CYP24A1), also mainly expressed in the kidney 2+ and 1,25(OH)2D3 are normal, 1,25(OH)2D3 inhibits its own synthesis by reducing CYP27B1 expression, while low serum levels of 1,25(OH)2D3 are often associated with a compensatory increase in CYP27B1 expression to restore homeostasis 2+ challenge. In fructose-fed lactating rats and in fructose-fed rat models of chronic kidney disease, both 1,25(OH)2D3 levels and CYP27B1 expression are low 2D3 and CYP27B1 levels, but also increased CYP24A1 expression, suggesting that high fructose intakes enhance the catabolism and impair the synthesis of 1,25(OH)2D32+ or some other mechanism could not be determined.The precursor vitamin D2D3 insufficiency independent of its effects on adaptive adjustments in Ca2+ transport. We first demonstrated that adult mice challenged with a low Ca2+ diet failed to upregulate Ca2+ transporter expression if fed a high 43% fructose diet for five wk, because fructose prevented compensatory increases in 1,25(OH)2D3 levels. To minimize potential diet-induced changes in Ca2+ transport, we then fed a second group of adult mice with normal Ca2+ but with a much higher (60%) fructose concentration for a longer duration of three mo, and eventually found the excessive fructose consumption did reduce levels of 1,25(OH)2D3 independent of Ca2+ transporter expression. Finally, we used rats to enable us to sample blood levels of 1,25(OH)2D3 and of other hormones, as well as of Ca2+ and Pi concentrations, each month and determine the time course of fructose effects on 1,25(OH)2D3.The aim of this study is to test the hypothesis that chronic fructose consumption causes 1,25(OH)All the procedures in this study were approved by the Institutional Animal Care and Use Committee, Rutgers, The State University of New Jersey.2+ transport and serum 1,25(OH)2D3 levels Study 1: mice fed fructose for five wk. Four-wk-old C57BL/6 mice , of similar initial body weights were randomly divided into four groups (n = 5) and pair-fed a 43% glucose or 43% fructose diet each containing normal (0.5%) or deficient Ca2+ (0.02%) levels . The levels of Ca2+ in the diet were based on previous studies Study 2: mice fed fructose for three mo. Four-wk-old mice were randomly divided into three groups (n = 5) and fed ad libitum a 63% glucose, 63% fructose, or 63% starch diet containing normal Ca2+ levels for three mo . The higher dietary fructose concentration and the longer feeding duration were designed to attempt to determine fructose effects on 1,25(OH)2D3 under conditions of Ca2+ sufficiency. Study 3: rats fed fructose for three mo. Four wk old rats were randomly divided into two groups (n = 6) fed a 43% glucose or 43% fructose diet containing normal Ca2+ levels ad libitum for three mo . Rats were utilized to ensure a large enough volume of serum would be collected each month for monitoring of Ca2+, 1,25(OH)2D3, phosphate (Pi), as well as the putative Pi and 1,25(OH)2D3 regulators, fibroblast growth factor 23 (FGF23) and parathyroid hormone (PTH).Experimental diets were modified from a standard American Institute of Nutrition (AIN)-93G formula by the manufacturer . Previous work indicated that 4 wk is sufficient for fructose consumption to have significant effects on intestinal Ca2+ transport rates at 37°C as described previously 2+ is localized 2+ transport buffer inner serosal and outer luminal compartments had equal initial concentrations (0.25 mM) of nonradioactive Ca2+, then a tracer concentration of 45Ca2+ was added to the outer mucosal compartment. After 1 h, the active accumulation of 45Ca2+ in the inner serosal compartment was calculated as a ratio of the final concentration of 45Ca2+ in compartments, inside/outside.Intestinal segments were everted immediately after isolation and prepared as sacs to determine Ca2+ concentrations were determined by previously described techniques using flame atomic absorption spectrophotometry  Following earlier work 2D3 levels were measured by enzyme immunoassay (ImmunoDiagnosticSystems (IDS), Arizona). Serum samples were de-lipidated by adding a solution containing dextran sulphate and magnesium chloride, and then centrifuged at 10000×g for 10 min. The resulting supernatant containing the de-lipidated serum is then 1,25(OH)2D3 immuno-extracted from potential cross-reactants by incubation for 90 minutes with a highly specific solid phase monoclonal anti-1,25(OH)2D3 before assaying by intact enzyme-linked immuno-sorbent assay (ELISA) for 1,25(OH)2D3 levels according to the manufacturer’s instructions Following earlier work Western blot analyses were performed using 50 μg of intestinal or renal protein extracts following earlier work EF1α) whose expression is independent of age and dietary carbohydrate  To examine the effects of animal age on expression of intestinal Ca2+ transporters and of renal 1,25(OH)2D3 metabolic enzymes, the glucose-fed mouse samples from Studies 1 and 2 were re-run together through real time RT-PCR with results normalized to the older, 4-mo-old, glucose-fed mice of study 2.Total RNA from homogenized intestinal mucosa or kidney was isolated, reverse transcribed, and real-time RT-PCR performed using Mx3000P  as previously described 2+ level, and of fructose and feeding time course, respectively. If an initial two-way ANOVA indicated a significant effect, a one-way ANOVA followed by LSD test  determined if differences existed between groups. Differences were considered significant at P<0.05. In study two, a one-way ANOVA was used to test for fructose effects.Data are presented as means ± SEM. For studies one and three, a two-way ANOVA was used to determine the effect of fructose and Ca2+ transport and transporter mRNA were associated with a low Ca2+ diet containing fructose as main carbohydrate source. Active transepithelial Ca2+ transport rate was modest in mice fed fructose-based, normal Ca2+ diet and was similar to that in mice fed a glucose-based, normal Ca2+ diet . As we previously demonstrated, intestinal GLUT5 expression increased with dietary fructose, regardless of dietary Ca2+ level , suggesting that the inhibitory effect of fructose on Ca2+ transporter expression is specific.Differences in intestinal Caa2+ diet Fig. 1A.2D3 were associated with fructose and low Ca2+ diets. In the kidney of mice fed low Ca2+, mRNA levels of CYP27B1 increased significantly by 17- and 7-fold, respectively for glucose- and fructose-based diets, compared to a normal Ca2+ diet with glucose or fructose as carbohydrate source e source Fig. 2A.2D3 was significantly increased with low Ca2+ 2D3 level which was not significantly different from those in mice fed normal Ca2+ diets (P>0.075 by one-way ANOVA). A low Ca2+ diet was associated with a significant 40% decrease in circulating level of FGF23, independent of dietary sugar concentrations. However, serum concentrations of Ca2+ and Pi which are primarily regulated by 1,25(OH)2D3 and FGF23, did not vary with diet.As expected, the circulating level of 1,25(OH)low Ca2+ Table 1 2+ transporters were associated with low Ca2+ diets. Low dietary Ca2+ induced a ∼two-fold increase in renal TRVP5 and CaBP9k mRNA levels in combination with either glucose or fructose . Duodenal Ca2+ transport rates did not vary with carbohydrate source and were similar in glucose-, fructose- and starch-fed mice  feeding Fig. 4A.2+ transport rates between 2D3 regulating enzymes in a direct comparison of mRNA expression levels in mice fed normal Ca2+ and glucose from study 1 (those mice were 2.5 mo old at sacrifice) and mice fed normal Ca2+ and glucose from study 2 (those mice were 4 mo old at sacrifice). Intestinal mRNA expression levels of TRPV6 and CaBP9k were both ∼100-fold lower at 4 than at 2.5 mo of age . Low CaBP9k expression at 4 mo-old mice fed normal Ca2+ explains the undetectable CaBP9k protein levels in these mice. Despite the large age-related differences in Ca2+ transport and transporter expression, expression of renal CYP27B1 and CYP24A1 was the same 2D3 levels. Rats fed a fructose or glucose diet displayed similar rates of food intake as well as similar final body weights . Similar to Study 2, the expression levels of intestinal and renal transporters with normal Ca2+ were also the same on fructose- and glucose-based diets 2D3 synthesizing renal enzyme, CYP27B1  diets , 7. Whil2D3 versus the glucose diet group 2D3 levels occur prior to increases in FGF23. Serum PTH, Ca2+ and Pi did not vary with duration or dietary sugar.As expected, the decreased CYP27B1 expression after three mo of consuming a high fructose diet was associated with a significantly lower circulating level of 1,25(OH)et group Table 2.2+ absorption and in 1,25(OH)2D3 levels, in response to dietary Ca2+ restriction. Thus, regardless of the source of Ca2+ challenge 2D3 levels independent of Ca2+ demand, and can alter CYP27B1 or CYP24A1 expression in the absence of physiological and nutritional Ca2+ challenge.We show in mice that excessive fructose intake inhibits adaptive increases in intestinal Ca2D3. This fructose-induced decrease in serum 1,25(OH)2D3 concentrations was not associated with a reduction in serum levels of its precursor 25(OH)D3, suggesting fructose had little effect on the initial steps of vitamin D synthesis. Instead, these findings indicate a specific effect of fructose at the renal step of either 1,25(OH)2D3 synthesis or degradation 2D3 levels was always associated with a fructose-induced decrease in CYP27B1 expression and less consistently with increased CYP24A1 expression. So far the effect of fructose on 1,25(OH)2D3 had been observed only when Ca2+ had been challenged. We demonstrate in the current study that chronic fructose intake reduces over time normal circulating levels of 1,25(OH)2D3 independently of any increase in Ca2+ requirement.Results from these current studies and our previous work 2+ absorption consists of a passive paracellular and a 1,25(OH)2D3-dependent active transcellular pathway 2+ is intestinal absorption, vertebrates undergoing Ca2+ stress adaptively increase active Ca2+ transcellular transport, achieved by augmenting levels of 1,25(OH)2D3. Here we confirmed in mouse our previous findings in rat models showing that marked increases in active absorption of Ca2+ are associated with equally remarkable increases in TRPV6 and CaBP9K expression, and that high fructose intakes dramatically compromise these adaptive increases in Ca2+ transport rates as well as in TRPV6 and CaBP9k mRNA or protein levels Total intestinal Ca2+ restricted conditions, the inhibition of adaptive increases in TRPV6 and CaBP9k expression by fructose likely explains the limited upregulation of active intestinal Ca2+transport in rodents with high fructose intakes. CaBP9k and TRPV6 had been shown to play a major role in the adaptation of intestinal Ca2+ transport under low Ca2+ conditions, and after induction by 1,25(OH)2D32+ is normal, TRPV6 null mice or CaBP9k null mice display a normal Ca2+ transport. However, in double KO TRPV6−/−/CaBP9k−/− mice, the adaptive increase in intestinal active Ca2+ transport in response to low Ca2+ diet or 1,25(OH)2D3 injection was reduced by 50% compared to that of wild-type mice, indicating that both CaBP9k and TRPV6 are needed for optimal Ca2+ transport under a Ca2+ challenge condition −/− mice 2+ absorption.In the present work, under Ca2+ stress and other workers have even used this gene for reference or “housekeeping” purposes 2+ transport across the apical membrane is the rate-limiting step We 2+ transport or Ca2+ transporter expression likely because active Ca2+ transport constitutes a very small component of total intestinal transport in older, Ca2+-sufficient animals. Comparing 2+ transport rate was also lower in older mice (study 2) which were fed normal Ca2+ for three mo than in younger mice fed the same sufficient level of dietary Ca2+ in Study 1. Compared to the 2.5 mo old mice, the modest rates of active Ca2+ transport rates exhibited by four mo old mice were also associated with reduced expression of TRPV6 and CaBP9K, as previously demonstrated in rats 2+ transport and transporter expression occurring in adult rodents between two and four mo of age may be linked to the age-related decrease in serum 1,25(OH)2D3 levels occurring simultaneously, as shown in this study and as previously demonstrated in rats 2D3 levels should be further investigated in mice on the same diets as a limitation of our age comparison is that different  diets were utilized.In four mo old mice and rats, fructose did not affect the active Ca2+ and Pi levels and not under Ca2+ stress have normal serum Ca2+ and Pi levels even when fed high fructose diets. The active, NaPi2b-mediated component of intestinal Pi transport also decreases dramatically in postweaning rodents as growth rate decreases markedly with age 2+ and Pi may be sufficient to maintain serum levels as demand for these minerals in older rodents is low.In this and in previous work, adult rats and mice fed sufficient Ca2D3 by inhibiting CYP27B1 expression and increasing CYP24A1 expression 2D3 or of Pi.FGF23 is a hormone synthesized mainly in the osteocytes and osteoblasts of bone and regulates blood levels of Pi via removal of Pi transporters from the apical membrane of kidney cells, thus reducing Pi reabsorption and increasing urinary excretion 2D3 levels 2+ diet and in rats fed for three mo a fructose-based, normal Ca2+ diet 2D3), FGF23 production did not increase with fructose feeding. In mice, the failure of fructose to induce FGF23 production may be due to the confounding effects of hypocalcaemia that acutely reduces circulating level of FGF23 2+ deficiency, circulating levels of FGF23 are low, thus allowing 1,25(OH)2D3 circulating levels to remain elevated and prevent decreases in blood Ca2+. In adult rats, the mechanism of the fructose-induced decrease in 1,25(OH)2D3 levels remains unclear, as fructose-induced increases in FGF23 levels were not significant perhaps due to a statistically insufficient number of samples. Fructose, however, is known to injure the kidneys 2D3 synthesis. PTH levels were not affected by fructose as shown in this as well as in our previous studies In a previous study using growing rats, we found that chronic consumption of fructose was associated with a significant increase in circulating levels of FGF23, which could potentially be the mechanism by which fructose reduces 1,25(OH)S3). Additionally, rodents and humans have different diets. The AIN recommends either 73% (AIN-93M (maintenance) diet) or 63% (AIN-93G (growth) diet) carbohydrate, levels higher than the average for humans (54%) consuming a high carbohydrate diet 2D3 levels 2D3, but only after a longer period of feeding (3 mo). Humans consume lower concentrations of fructose that are often mixed with other carbohydrates, but studies have shown that mixing with glucose may accelerate fructose effects. Because of the cumulative effects of fructose demonstrated here, lower fructose concentrations will likely cause similar deleterious effects as higher fructose concentrations if consumed for a longer time period There are limitations to this study, both in conduct and extrapolation of the results to the human condition. Despite similar food intakes, fructose feeding was associated with a significantly greater kidney mass index (kidney weight/body weight) in both the longer-term studies (two and three) in both mice and rats Figure S12+. AThe body and kidney weight of mice pair-fed diets containing either 43% glucose or fructose, in combination with either normal or low Ca) Initial and final body weight after 5 wk of feeding on the experimental diets. B) Kidney weight after 5 wk. C) Kidney somatic index. Bars are means ± SEM; n = 5–8. Nor  =  normal; G  =  glucose; F  =  fructose. Differences (P<0.05) among means are indicated by differences in superscript letters, as analyzed by 1-way ANOVA LSD. Thus, bars with superscript “a” are > bars with “b” and similar to those with “ab”.(TIF)Click here for additional data file.Figure S22+ and 63% glucose, fructose or starch. AThe body weight, feeding rate, and kidney weight of mice fed diets containing normal Ca) Body weight after 3 mo of feeding on the special diets. B) Feeding rate per day normalized to body weight. C) Kidney weight after 3 mo of feeding on the special diets. D) Kidney somatic index. Bars are means ± SEM; n = 5–8. Differences (P<0.05) among means are indicated by differences in superscript letters, as analyzed by 1-way ANOVA LSD. Thus, bars with superscript “a” are > bars with “b”.(TIF)Click here for additional data file.Figure S3The body and kidney weight of rat fed diets containing either 43% glucose or fructose. A) Body weight after 3 mo of feeding of the special diets. B) Feeding rate per day normalized to body weight. C) Kidney weight after 3 mo of feeding of the special diets. D) Kidney somatic index. Bars are means ± SEM; n = 5–8. Differences (P<0.05) among means are indicated by differences in superscript letters, as analyzed by 1-way ANOVA LSD. Thus, bars with superscript “a” are > bars with “b”.(TIF)Click here for additional data file.Figure S4CaBP9k protein abundance. The protein abundance of CaBP9k was determined in the small intestine of mice fed diets containing either 43% glucose or fructose, in combination with either normal or low Ca2+. β-actin was used as a reference. Nor  =  normal; G  =  glucose; F  =  fructose.(TIF)Click here for additional data file.Figure S5mRNA levels of GLUT5. Expression level of GLUT5 in the intestine of A) mice fed diets containing either 43% glucose or fructose, in combination with either normal or low Ca2+ for 5 wk; B) mice fed diets containing normal Ca2+ in combination with 63% glucose, fructose or starch for 3 mo; and C) rats fed diets containing either 43% glucose or fructose for 3 mo. All expression data were analyzed by real-time PCR using EF1α as a reference and normalized relative to levels seen in rat fed glucose diet. Nor  =  normal; G  =  glucose; F  =  fructose. Data are means ± SEM (n = 6 per group). Differences (P<0.05) among means are indicated by differences in superscript letters, as analyzed by 1-way ANOVA LSD. Thus, for each study, bars with superscript “a” are > bars with “b”.(TIF)Click here for additional data file.Table S1Primer sequences.(DOC)Click here for additional data file.


Myofibroblast contraction is fundamental in the excessive tissue remodeling that is characteristic of fibrotic tissue contractures. Tissue remodeling during development of fibrosis leads to gradually increasing stiffness of the extracellular matrix. We propose that this increased stiffness positively feeds back on the contractile activities of myofibroblasts. We have previously shown that cycles of contraction directly correlate with periodic intracellular calcium oscillations in cultured myofibroblasts. We analyze cytosolic calcium dynamics using fluorescent calcium indicators to evaluate the possible impact of mechanical stress on myofibroblast contractile activity. To modulate extracellular mechanics, we seeded primary rat subcutaneous myofibroblasts on silicone substrates and into collagen gels of different elastic modulus. We modulated cell stress by cell growth on differently adhesive culture substrates, by restricting cell spreading area on micro-printed adhesive islands, and depolymerizing actin with Cytochalasin D. In general, calcium oscillation frequencies in myofibroblasts increased with increasing mechanical challenge. These results provide new insight on how changing mechanical conditions for myofibroblasts are encoded in calcium oscillations and possibly explain how reparative cells adapt their contractile behavior to the stresses occurring in normal and pathological tissue repair. Myofibroblasts play a beneficial role during normal tissue repair by synthesizing, contracting and remodeling the extracellular matrix (ECM) 2+]i) 2+]i occur in cultured fibroblasts and myofibroblasts and are directly correlated with subcellular contractile events measured with the atomic force microscope 2+]i oscillations in low contractile cardiac and subcutaneous fibroblasts is longer than in their highly contractile myofibroblast counterparts 2+]i oscillatory activity of myofibroblasts regulating subcellular contractions has not been studied.We have recently provided experimental evidence that myofibroblasts employ two modes of contraction, acting simultaneously but independently in a lock-step mechanism 2+]i oscillation frequency. Using [Ca2+]i oscillations as an indicator, we analyze the contractile activity of rat subcutaneous myofibroblasts (SCMF) as a function of the mechanics in different cell culture models. We modulate ECM stiffness by seeding SCMF onto two-dimensional silicone culture substrates and into three-dimensional collagen gels of increasing E-modulus. We vary intracellular stress by modifying cell adhesion strength through different surface coating, by restricting cell size by growing myofibroblasts on specific surface areas created by microcontact printing (μCP), and by inhibiting actin polymerization. Our results demonstrate that increasing mechanical stress increases the frequency of [Ca2+]i oscillations in SCMF. Our findings help to understand how changing mechanical conditions for myofibroblasts are encoded in calcium oscillations and possibly explain how reparative cells adapt their contractile behavior to the changing stresses in pathological tissue repair.We hypothesize that the static mechanical conditions of the extracellular environment control the contractile remodelling activity of myofibroblasts by modulating i oscillations with 50 nM endothelin-1 , as growth on isolated islands reduced spontaneous [Ca2+]i oscillations. A series of independent experiments indicated that the lower cell density and larger spacing between cells was causal for the lack of spontaneous oscillations on square islands (data not shown). Third, we inhibited actin polymerization using Cytochalasin D .To modify intracellular stress, we first seeded SCMF onto coverslips coated with 2 μg/cm2+]i changes, we incubated cells for 45 min with 3 μM Fura-2 AM (Life Technologies) in F-12 medium containing 10% FBS, 20 mM HEPES and 0.25% pluronic acid (Sigma-Aldrich). When plastic coverslips were used as substrate, we incubated cells with 20 μM Fluo-4 AM (Life Technologies) since plastic is autofluorescent at the excitation wavelengths of Fura-2. Next, we washed cells for 15 min with F-12 medium containing 10% FBS and 20 mM HEPES, before recording [Ca2+]i dynamics at 37°C on an inverted microscope  in a humidified atmosphere. The microscope was equipped with a polychromatic Xenon light source , a high numerical aperture objective  and a charge-coupled device camera .To visualize [Ca2+]i were calculated over regions of interest covering the entire cells and expressed in arbitrary units as fluorescence ratio  =  Em340/Em380, as defined in 2+ indicator, excitation and emission wavelengths were 488 nm and 515 nm, respectively. Fluo-4 fluorescence ratio was expressed by F/F0 (fluorescence/fluorescence at rest). We calculated the mean period of regular [Ca2+]i oscillations and we compiled data of each group into histograms. Distributions were fitted using MatLab software  and we verified the goodness of the fit with the EasyFit software (MathWave Technologies). For experiments performed with Cytochalasin D, we recorded fluorescence for two 15 min bouts intercalated by a 15 min period during which we added the drug and allowed it to act. We compared [Ca2+]i oscillations before and after addition of Cytochalasin D for the same cell.Cells incubated with Fura-2 were successively excited at 340 nm and 380 nm every 5 s and pairs of frames were recorded at 510 nm for 15 min, using OpenLab 3.0.6 software . Changes in i oscillations in myofibroblasts, we first seeded SCMF on silicone substrates of different E-moduli. Substrate E-moduli were tuned to model the mechanical aspects of connective tissues that are either soft/normal (5 kPa), under repair (15 kPa) or fibrotic/stiff (50 kPa) 2+]i indicators. Changes in fluorescence over time, representing spontaneous [Ca2+]i oscillations in single cells were recorded (2+]i peaks/min), increased with increasing substrate E-modulus , or 5.0 μg/cm2 PLL (low adhesive) 2+]i profiles were analyzed for SCMF , 0.5 μg/cmfor SCMF , we obta/cm2 PLL . A numbe/cm2 PLL , the avem2 on FN . Similarm2 on FN . Using fquencies .2 and large islands of 4,900 µm2 to perform [Ca2+]i oscillation experiments . Because isolated growth on spaced islands prevented spontaneous [Ca2+]i oscillations in SCMF, we provoked [Ca2+]i oscillations using 50 nM ET-1 in these experiments only . Hence, destruction of the actin cytoskeleton led to an overall decrease in the [Ca2+]i oscillation activity of SCMF.Finally, we assessed the role of an intact actin cytoskeleton in regulating i peaks are directly followed by subcellular contractile events in myofibroblasts 2+]i oscillation periods as an indicator for the contractile activity of SCMF in response to varying extracellular and intracellular mechanical stress. We demonstrate that growing SCMF on silicone substrates and in collagen gels of increasing E-modulus, as well as augmenting intracellular stress by manipulating cell adhesion and spreading all increase the [Ca2+]i oscillation frequency. Conversely, eliminating stress by disrupting actin stress fibers resulted in the reduction of [Ca2+]i oscillation frequencies. Collectively, our data demonstrate that the level of extracellular mechanical challenge modulates oscillatory [Ca2+]i dynamics and may thus control the subcellular contractile activity of myofibroblasts.Myofibroblast contraction contributes to normal wound healing and is instrumental in the development of fibrotic contractures 2+ is an important second messenger in transducing mechanical signals. A number of reports have demonstrated Ca2+ influx over locally and globally stretched fibroblast membranes  are in the same range as those reported for cardiac myofibroblasts cultured on 15 kPa soft silicone substrates (∼90 s), and SCMF (∼50 s) and subcutaneous fibroblasts (∼70 s) cultured on glass 2+]i peaks in myofibroblasts regulate periodic contractile events 2+]i-regulated contractions is expected to result in incremental ECM remodelling in a ‘lock-step’ or ‘ratchet’ mechanism 2+]i oscillation frequencies in conditions of higher ECM stiffness will result in more efficient ECM remodelling. Together with the finding of other studies that fibroblasts generate higher isometric forces on stiff than on soft substrates Overall, SCMF developing larger focal adhesions, more pronounced stress fibers and higher intracellular stress always displayed faster [Cain vitro conditions of poor healing in vivo can be related to the modulation of myofibroblast contraction at the single-cell and subcellular level and whether this effect is due to modulation of the calcium regulation pathway remain to be shown.Adaptation of myofibroblast contraction to the level of extracellularly imposed stress is physiologically relevant during tissue repair, when remodeling of the ECM by myofibroblasts generates a gradual increase of cell tension over time


The sensitivity of the method is calculated numerically for two useful dye pairs. As illustrative application of the enhanced measurement, we use fluo-4 and fluo-4FF to image the calcium wave produced by a cardiac myocyte in response to a small artificial calcium spark.We extend the sensitivity of quantitative concentration imaging to an approximately 1000-fold range of concentrations by a method that uses two fluorescent dyes with the same fluorophore, having different affinity for the monitored species. While the formulation and illustration refer to a monitor of calcium concentration, the method is applicable to any species that binds to multiple indicators with the same spectral properties. The use of a common fluorophore has the virtue of leaving vast regions of the electromagnetic spectrum available for other applications. We provide the exact analytic expression relating measured fluorescence to [Ca Optical sensors of concentration of a chemical species typically yield a signal  that grows with the concentration of the indicator bound to the monitored species. Therefore, the signals are limited at low concentrations by the sensitivity and at high concentration by the saturation of the sensor.2+]. The procedure was to apply as stimuli synthetic local increases in [Ca2+]  2+ from cellular stores. The applied artificial sparks could be varied in a wide range, from nM to tens of µM, while the cellular responses, the subject of the investigation, were also expected to vary widely. Many other situations can be envisioned that require the ability to monitor precisely the concentration of Ca2+ or other species over several orders of magnitude.Research in cell physiology often calls for dynamic measurement in ranges wider than can be handled by a single monitor. Specifically, we found such need while exploring the response of cardiac and skeletal muscle cells to imposed increases in free  the following equations apply:F isCaD and CaE are concentrations, and mj and Mj represent the molar fluorescence of dye j in its free and Ca2+-bound forms.For the derivation we assume that the aqueous solution  contains a CaF is a single-valued, monotonically increasing function of [Ca2+]. An explicit representation of it can be derived from 2+] from F, as followsCaD and CaE can also be derived from 10 [Ca2+] vs. f, which is F normalized to its maximum (DTMD+ETME), for fluo-4 and fluo-4FF used at equal concentrations. For comparison the same function for fluo-4FF alone is plotted in black. The dependence is in this caseR is the dynamic range, ME/mE, of dye E.2+], rather than its log, is used in the definition, S will be maximal at [Ca2+] = 0 and decrease monotonically with increasing dye occupancy. In red is the sensitivity, defined again by function (6), for the two-dye case of fluo-4 and fluo-4FF. Given that the dissociation constants are 0.45 and 9.7 µM, the range of high sensitivity is extended to greater than two orders of magnitude. For an additional comparison we plotted in green trace the sensitivity attained with equal concentrations of fluo-4 and the low affinity dye fluo-5N.The purpose of using two dyes is to extend the range of concentrations that can be measured with high sensitivity. The sensitivity of the dual dye method is compared with that of a single dye in In the representation of 2+] to dye-Ca2+ concentrations without assuming equilibrium isThe derivation above assumed equilibrium between both dyes and calcium. The assumption will not be valid when the goal is to monitor rapid changes with high affinity dyes. The equation that relates , CaD and CaE. The expression for [Ca2+] isThe system of CaD and CaE with kinetic corrections, are also available to the interested reader.Analytical expressions of the concentrations 2+] in a rabbit atrial cardiomyocyte, derived from fluorescence of fluo-4 and fluo-4FF. The cell, whose plasmalemma was permeabilized with saponin, was immersed in an internal solution with known concentrations of both dyes and was stimulated by a small artificial Ca2+ spark. The spark was produced by two-photon lysis of the Ca2+ cage NP-EGTA, present at a concentration of 0.4 mM. Photolytic irradiation and image acquisition were carried out with a dual confocal scanner .As an example The resting fluorescence of the cell is represented in the first frame . The artificial spark was produced outside the cell by brief irradiation at a diffraction-limited spot 3 µm away from the plasmalemma (target in first frame) with IR light pulsed for two-photon excitation. Images were acquired at one frame every 1.8 ms. The figure shows one every five images of this set .2+ release from the sarcoplasmic reticulum, results in a propagating Ca2+ wave. The extended range of the two-dye technique allows for accurate, fast, nearly linear monitoring of the wave and precise imaging of the artificial spark. Note in particular the “trigger [Ca2+]” measured at the point where Ca2+ in the artificial spark reaches the cell membrane, at a time just before the first hint of a response (the frame at 90 ms). This trigger [Ca2+], 240 nM in the example, was just 50 nM above the resting [Ca2+] level. By systematically varying the intensity and duration of IR light, trigger [Ca2+] could be varied to find a minimum trigger, or “threshold [Ca2+]”, which could be defined with a precision of 10 nM. The ability to measure [Ca2+] in an extended range with a single fluorophore is being used to advantage in current applications, in which we add imaging of [Ca2+]SR by SEER of fluorescence of mag-indo-1 The cellular response, consisting in CaSome limitations in the applicability of the technique should be acknowledged. The use of two dyes of different affinity extends the measurable range of concentrations but cannot be construed as providing an extended range where linearity between signal and concentration applies. The linear range is limited to that of the low affinity dye. If the two affinities are very different there may be an additional approximately linear range, at concentrations where the high affinity dye is saturated.2+] with The calculation of  of 190 nM. A diffraction-limited spot 3 µm away from the plasmalemma was irradiated for 1 ms with IR light of 710 nm pulsed for two-photon excitation. Images were acquired at one frame every 1.8 ms. [Ca2+] was derived from fluorescence according to DT and ET were known in the applied solution. DT and ET inside the cell were calculated according to resting fluorescence, assuming that [Ca2+], MD, mD, ME and mE in cytosol were the same as in the solution, and that both dyes bound equally inside the cell. The molar fluorescence parameters were determined separately in solution for both dyes. Additional details of solution composition, perfusion, irradiation and image acquisition and processing were provided previously Briefly, animals were anesthetized with thiopental sodium . After thoracotomy, hearts were excised, mounted on a Langendorff apparatus, and retrogradely perfused via the aorta with Liberase Blendzyme TH containing solution (37°C). Myocytes were used for experimentation within 1–6 hours after isolation. Myocytes adhered to the coverslip bottom of a custom chamber on the stage of a microscope, where its plasma membrane was permeabilized by saponin treatment (0.002% for 2 min). Photolytic irradiation and simultaneous image acquisition were carried out with a dual confocal scanner ) combining a slit scanner (LSM 5


G0/G1 phase arrest through the effects of associated protein such as p53, p21, p17, CDK2 and cyclin E, and it triggered apoptosis through promotion of Bax and Bid expression and reduction of Bcl-2, leading to decrease the levels of mitochondrial membrane potential (ΔΨm), and followed the releases of cytochrome c, AIF and Endo G then for causing apoptosis in HSC-3 cells. These results suggest that PEITC could be an antitumor compound for oral cancer therapy.Phenethyl isothiocyanate (PEITC), an effective anticancer and chemopreventive agent, has been reported to inhibit cancer cell growth through cell-cycle arrest and induction of apoptotic events in various human cancer cells models. However, whether PEITC inhibits human oral squamous cell carcinoma HSC-3 cell growth and its underlying mechanisms is still not well elucidated. In the present study, we evaluated the inhibitory effects of PEITC in HSC-3 cells and examined PEITC-modulated cell-cycle arrest and apoptosis. The contrast-phase and flow cytometric assays were used for examining cell morphological changes and viability, respectively. The changes of cell-cycle and apoptosis-associated protein levels were determined utilizing Western blotting in HSC-3 cells after exposure to PEITC. Our results indicated that PEITC effectively inhibited the HSC-3 cells' growth and caused apoptosis. PEITC induced  The major contributors for oral cancer include tobacco and alcohol consumption , 2 as wem) which released cytochrome c and thereafter complexes with apoptotic protease activating factor-1 (Apaf-1) to form apoptosome activates caspase-9 and caspase-3, leading to apoptosis -2,7-dichloro-3-oxo-3H-xanthen-9-yl]-2-[2-[2-[bis [2-[(acetyloxy)methoxy]-2-oxyethyl]amino]-5-methylphenoxy]ethoxy]phenyl]-N-[2-[(acetyloxy)methoxy]-2-oxyethyl]-, (acetyloxy)methyl ester (Fluo-3/AM) and 3,3′-dihexyloxacarbocyanine iodide . Cells were maintained in DMEM/F-12 (1 : 1) containing 10% FBS, 2 mM L-glutamine, 100 Units/mL penicillin and 100 5) were placed in 12-well plates, and then PEITC was added to each well at final concentrations of 0.5, 1, 2, 2.5 and 5 μM. DMSO at 0.5% in media served as a vehicle control. Cells were incubated for 24, 48 and 72 h. For morphological changes, cells in each well were examined for 24 and 48 h and photographed under a phase-contrast microscope . They were then detected using the ECL kit (Millipore) and autoradiography using X-ray film [β-actin antibody for ensuring that equal proteins were loaded [HSC-3 cells  , 28. Theray film , 28. Eace loaded , 28. TheThe score functions in the Discovery Studio 2.5 which we used to predict the protein active binding pockets and simulate the possible ligand poses to present the interaction forces between the cdc25A (protein) and PEITC (ligand). There are two major parts of the LigandFit  docking:in AMBER . Insteadt-test, a probability of P < 0.05 being considered significant.The results are presented as mean ± S.E.M, and the difference between the PEITC-treated and control groups was analyzed by Student's μM) for 24, 48, or 72 h. The cell-morphological changes were examined and photographed by a phase-contrast microscope. The results shown in Figures μM of PEITC. Furthermore, the percentage of viable cells was measured by flow cytometric assay. Then cells were treated with various doses of PEITC for 24, 48, and 72 h, and PEITC decreased the percentage of viable cells in a dose- and time-dependent manner  the PEITC treatment decreased the percentage of viable cells and induced cell morphological changes in a dose-dependent manner; (2) PEITC induced apoptosis in a time-dependent manner; (3) PEITC promoted the production of ROS and Ca2+ but decreased the levels of ΔΨm in a time-dependent manners; (4) PEITC increased the proapoptotic protein Bax and decreased the antiapoptotic protein Bcl-2, both proteins involved the levels of ΔΨm for cell to survive or apoptosis [Other investigators already pointed out that ROS might be fully involved in cellular responses to PEITC , 32, andpoptosis .G0/G1 phase arrest in HSC-3 cells. Thus, PEITC induced downregulation of cyclins and CDKs and upregulation of the CDK inhibitor p21 expression, whereas upregulation of p27 for causing G0/G1 arrest. In this regard, direct covalent modification of cellular proteins has been suggested to be an important early event in the induction of cell-cycle arrest by PEITC [1 of the cell cycle and entry into the S phase, which are predominantly cyclin D1/CDK4 and cyclin E/CDK2 [2/M arrest in PC-3 human prostate cancer cells [2/M arrest was associated with proteasome-mediated degradation of CDK1 and cdc25c [G0/G1 phase arrest in HT29 human colon cancer cells [G0/G1 phase arrest, we found that PEITC treatment caused a decrease of S-phase cells in HSC-3 cells. Thus, the different cell lines which may have different responses are not clear at present study. Apparently, further investigations are needed.Furthermore, our results also show that PEITC decreased expression of cdc25A, CDK6 and cyclin D , CDK2 anby PEITC . It is wby PEITC –36. The n E/CDK2 ; moreoven E/CDK2 , 37. Hower cells  and HL-6er cells . It has d cdc25c . Howeverer cells . In addic release and caspase-3 activation by certain apoptotic stimuli such as hyperoxia [c in some cellular models of mitochondria-mediated apoptosis [m and cytochrome c release, the activation of caspase-9 and caspase-3 (It was reported that ROS had function upstream of cytochrome yperoxia  and the poptosis . Here, waspase-3  for causaspase-3 , leadingaspase-3  and mitoaspase-3 . Hydrogeaspase-3 .2+ production that caused ER stress which based on increasing the GRP78 and ROS, then led to the changes of Bax/Bcl-2 for causing the decreased of ΔΨm and then led to cytochrome c, AIF and Endo G release, then to activate caspase-9 and -3 for causing apoptosis.In conclusion, PEITC induced apoptosis in HSC-3 cells which are summarized in


L, FLIPS and FLIPR) induced pro-apoptotic responses to TNF, with FLIPL knock-down providing the greatest apoptotic switch. However, FLIPL knock-down consequently increased expression of HO-1; a response that occurred in AML (but not non-cancerous) cells to protect a proportion of them from apoptotic death. Our results show that increases in HO-1 induced an apoptotic-resistant form in AML cells in the absence of FLIPL. This is the first time that FLIPL has been shown to regulate the expression of HO-1. These data reveal unique regulatory networks in cancerous AML cells whereby FLIP regulation of HO-1 provides AML cells with secondary anti-apoptotic protection against extrinsic factors (eg TNF/chemotherapies) that try to switch on death signals in these highly death-resistant cells. Future AML therapies should target these mechanisms.Acute myeloid leukemia (AML) comprises a heterogeneous group of clonal disorders of hematopoietic progenitors. We previously showed that heme oxygenase-1 (HO- 1/Hsp32) underlies resistance of AML to TNF-induced apoptosis. Here we show for the first time that the modulatory protein, FLICE-inhibitory protein (FLIP) indirectly regulates induction of HO-1 in response to TNF in human AML blasts, but not noncancerous control cells. In AML cells, TNF-induced FLIP expression was an NF-κB-dependent event, and silencing of FLIP isoforms (FLIP Acute myeloid leukemia (AML) is a malignancy of the myeloid progenitors, characterized by the rapid growth of abnormal myeloid cells that accumulate in the bone marrow and blood. AML is the most common acute leukemia affecting adults, and its incidence increases with age with 75% of patients diagnosed after the age of 60 . FurtherApoptosis is an essential part of mechanisms that maintain normal cellular homeostasis. Deregulation of the apoptosis machinery and evasion of apoptosis is a mechanism of most human malignancies, including AML ,4. Most L, FLIPS and FLIPR)[S and FLIPR have been shown to have a clear anti-apoptotic function, the role of FLIPL in regulating apoptosis has been found to be quite controversial [L as an anti-apoptotic molecule that blocks caspase activation, whereas, others suggest that FLIPL catalyzes caspase-8 processing at the DISC and therefore has a pro-apoptotic function.FLIP mediated regulation of the DISC is through the modulation of activation of procaspase-8 and thereby prevents induction of apoptosis mediated by death receptors . The humnd FLIPR). The FLIoversial ,11. MostL is induced in a NF-κB-dependent response [L had only a limited role in regulating apoptosis in AML cells in response to TNF-mediated death responses. Here we demonstrate a novel pathway involving FLIPL negatively regulating the expression of heme oxygenase-1 (HO-1), whose role in chemoresistance, both in AML and other cancers, is well established.We have previously shown that in the human AML cell line THP-1, FLIPresponse . In the L, is induced in AML cell line THP-1 and HL60, in response to TNF in a NF-κB-dependent mechanism [L FLIPS and FLIPR) has not been wholly established in human AML. Therefore, we wanted to fully elucidate the induction of FLIP isoforms (FLIPL FLIPS and FLIPR) in response to TNF in human AML patient samples, AML cell lines and non-malignant control cells. Cells were treated with TNF for various time points Figure L, FLIPS and FLIPR are all induced by TNF in a time dependent fashion, with all FLIP isoforms mRNAs being significantly raised from at least 4 h treatment with TNF. FLIPL has the greatest induction in all cells tested suggesting that this protein may have the most influence on apoptotic pathways. Figure L and FLIPS, but not FLIPR by TNF is a NF-κB-dependent response. The slight but significant mRNA induction of FLIPR by TNF is not controlled by NF-κB. Figure R, thus suggesting that mRNA is not processed into protein or the protein level of FLIPR is lower than the detectable concentration for this assay.Previously we have shown that the FLIP isoform, FLIPechanism . Others echanism . MoreoveL, FLIPS and FLIPR expression in human AML we used siRNA to target knockdown of these modulators. Figure L, FLIPS and FLIPR in human AML cells. When we knocked down FLIPL, FLIPS and FLIPR expression in AML cell line THP-1 and human monocytes  of AML cells to become sensitive to TNF-induced death, compared to only 6% of the same cells dying in the absence of TNF. Interestingly, knockdown of HO-1 alone allowed only 15% of AML cells to be sensitive towards TNF-induced death. These findings suggest that together FLIPL and HO-1 protect AML cells from TNF-induced apoptosis and both need to be targeted for effective AML therapy.Finally we wanted to determine if silencing FLIP1 Figure  we showeL can regulate the expression of HO-1, and that targeting knockdown of both molecules using siRNA improves apoptosis in response to TNF treatment. Most research to date regarding the importance of FLIP in regulating cell survival has focused on its role in preventing the formation of the DISC. This is the first time FLIP has been shown to regulate HO-1 gene induction in human cells, moreover, targeting both HO-1 and FLIP in combination could markedly improve survival rates in human AML.Understanding host responses that modulate apoptosis in human AML is key to developing new therapeutic strategies to reduce the mortality associated with this complex disease. The present study demonstrates that FLIPL which is induced in AML in response to TNF in a NF-κB-dependent manner, is able to inhibit apoptosis by modulating activation of procaspase-8 and thereby inhibiting formation of the DISC [L but not FLIPS or FLIPR, induced the expression of the cytoprotective protein HO-1.The regulation of apoptosis in human AML is the focus of our research. We have previously shown that inhibiting HO-1 and NF-κB in combination allows AML cells to undergo apoptosis, thus suggesting that both these pathways are needed for the survival of these malignant cells . HO-1 prthe DISC . We founL-targeted knockdown compared to control cells, is that removing FLIPL reduces the active NF-κB levels in human AML cells, thus removing its negative influence on the HO-1 promoter. This would allow HO-1 levels to come back to normal non-malignant control levels that are observed in ordinary hematopoietic stem cells (HSC).Expression of HO-1 is usually increased in human cancer cells, however we have recently shown that in human AML cells HO-1 expression is significantly lower when compared with nonmalignant control cells . FurtherL. However when we remove NF-κB activation using BAY-11-7082, a secondary antiapoptotic pathway regulated by the transcription factor Nrf2, becomes apparent and drives the induction of cytoprotective gene HO-1. In this study we show that we do get some TNF-induced apoptosis of AML cells when we knockdown FLIPL using siRNA, however when we combine the treatment to targeting HO-1 as well, we get a markedly increased level of apoptosis in response to TNF. This suggests that both FLIPL and HO-1 are two genes that when individually targeted have little or no effect on apoptosis of AML cells, but targeting these two genes in combination would provide a novel mechanism that future more successful therapies must employ to effectively treat AML.We have previously shown that AML cells protect themselves from TNF-induced apoptotic responses by two interconnected mechanisms. The first is the induction of NF-κB-driven anti-apoptotic gene FLIPL and HO-1 together have a role in controlling TNF-induced apoptotic responses in AML cells. It is also clear that knockdown of FLIPL induces HO-1 expression in human AML, but not corresponding non-malignant control cells The role of the other FLIP isoforms, FLIPS and FLIPR, is clearly less critical in AML cells in response to TNF. Moreover, the fact that we were unable to detect FLIPR protein makes it even less of a target molecule for future anti-AML therapy. Previously we have shown that HO-1 may play an important secondary role in protecting AML cells from apoptotic responses, here we cement the concept that HO-1 is indeed very important for the survival of AML cells and therefore should be targeted with new SACT.Our results clearly indicate that FLIPPrimary AML cells were obtained under local ethical approval (LREC ref 07/H0310/146). Informed consent was obtained from patients and any identifying information was anonymised according to the approved ethics code of practice.L, FLIPS and FLIPR siRNA were purchased from Applied Biosystems . All other reagents were obtained from Sigma-Aldrich , unless indicated.The AML-derived cell lines THP-1 and HL60 were obtained from the European Collection of Cell Cultures. Anti-human HO-1 antibody was purchased from Assay Designs . Anti-human FLIP antibody was purchased from Abcam . Control, HO-1, FLIP6/mL) were incubated in complete medium for 2 h at 37°C to allow adherence of monocytes. Positive selection of human hematopoietic stem cells (HSC) were isolated from PBMCs using a CD34 positive selection kit . For all CD34+ and primary monocyte experiments at least three different donors were used to obtain the results presented in this paper. AML samples that were less than 80% blasts and expressed CD34, were purified using the CD34 positive selection kit. Cell type was confirmed by microscopy and flow cytometry.For primary cell isolation, heparinized blood was collected from volunteers and human peripheral blood mononuclear cells (PBMCs) isolated by Histopaque (Sigma-Aldrich) density gradient centrifugation. PBMCs  core kit (Applied Biosystems). Real-time PCR primers were purchased from Invitrogen. Relative quantitative real-time PCR used SYBR green technology (Roche) on cDNA generated from the reverse transcription of purified RNA. After preamplification (95°C for 2 min), the PCRs were amplified for 45 cycles (95°C for 15 s and 60°C for 10 s and 72°C for 10 s) on a LightCycler 480 (Roche). mRNA expression was normalized against GAPDH expression using the standard curve method.Total RNA was extracted from 5 x 10SDS-PAGE and Western analyses were performed as described previously . Brieflyt test was performed to assess statistical significance from controls. Results with P < 0.05 were considered statistically significant (*). Results represent the mean ± SEM of 3 independent experiments. For Western blotting experiments, data are representative of at least 3 separate experiments.Student's


Further study of the biochemical mode of action of 1 has shown that it acts through a complicated mechanism in which the compound forms a covalent but reversible complex with MDMX and locks MDMX into a conformation that is unable to bind p53. The relative stability of this complex is influenced by many factors including the reducing potential of the media, the presence of aggregates, and other factors that influence the conformational stability of the protein. This complex mechanism of action hinders the further development of compound 1 as a selective MDMX inhibitor.SJ-172550 ( The p53 pathway is inactivated in virtually every human cancer by mutations in the p53 gene itself or other genes in the pathway 1) was identified in a high-throughput screen using a biochemical assay to recapitulate the binding of MDMX and p53 50 ∼ 5 µM and caused p53 dependent cell death of retinoblastoma cells 50 ∼ 30 µM in vitro and in vivoMore recently, small molecule inhibitors of MDMX have been described. The first . Indeed compound 1 will form adducts with glutathione  Compound 1 only appeared to function under non-reducing conditions; and 2) allowing MDMX and compound 1 to react in neutral pH acetate buffer gave clear covalent adducts detectable by mass spectrometry  under reducing conditions the majority of MDMX adopts a conformation that is competent to bind p53 and relatively non-reactive with compound 1. Additionally, compound 1 appears to shift the MDMX conformational equilibrium towards the conformation unable to bind p53 by reversible alkylation of Cys76. This complex, multi-mode mechanism greatly complicates the interpretation of experiments using 1 and limits its value as a lead compound for further development as a selective MDMX inhibitor.Therefore, we carefully analyzed the molecular mechanism of action. These studies led to three major findings: 1) MDMX exists as an ensemble of conformations that differ in both their ability to bind p53 and their reactivity with alkylating agents; 2) in non-reducing conditions or high inhibitor and MDMX concentrations, the majority of MDMX adopts a conformation that fails to bind p53 and is sensitive to alkylation - under these conditions 1 and MDMX failed to yield detectable adducts 1 and MDMX revealed clear adduct peaks . When high concentrations of compound 1 were allowed to react with either construct of the protein , and injected with minimal manipulation, adducts clearly formed, with either multiple alkylation events (GST-tagged protein) or a single alkylation event (untagged protein). These constructs were stable enough to at least partially survive a rapid desalting procedure prior to injection in the mass spectrometer. In the case of the untagged protein this stoichiometry afforded only partial labeling, which may reflect either low reactivity, or rapid reversal during desalting, or both. Thus, clearly compound 1 is capable of alkylating the single cysteine present in the p53 binding domain of MDMX. However, under these conditions the compound is not fully soluble (solubility of 12 µM in aqueous buffer) and exists mostly in aggregated form, which we have previously shown can result in aberrant protein behavior. In our original report, mass spectrometry following dialysis of a mixture of compound dialysis Figure 21, the reactions were also carried out at conditions more closely resembling those used in the original report . Under these conditions the binding of p53 peptide to MDMX is fully inhibited at equilibrium. In order to provide enough protein to give strong signal in the mass spectrometer, this mixture had to be rapidly concentrated (5 minutes) prior to desalting. No adducts were detected for either protein construct. One can conclude from these experiments and the data published in our original report 1 with MDMX is covalent but reversible.In order to test whether aggregation was a driver for the observed activity of compound 1 independent of construct and in a wide variety of conditions . These experiments suggest that the binding of compound 1 to MDMX utilizes both covalent and non-covalent interactions. Most likely this takes the form of pre-organization of the compound with the “active site” cysteine, followed by formation of a covalent adduct, which we have previously reported with other cysteine reactive electrophilic inhibitors Careful consideration of these results suggests that compound c center Figure 3Figure S6) is inhibited by compound 1 with 10-fold weaker potency than that seen with the MDM2 while the p53 binding potency of the mutant remains the same as that of the wild type MDM2. This reinforces the key concept outlined above – the interaction between compound 1 and MDMX/2 involves both covalent and non-covalent components with both being required for maximal potency. Moreover, the covalent component requires the presence of both the unsaturated functionality of compound 1 and the cysteine of the protein. The most likely mechanism is outlined in The hypothesis also implies that the interaction requires the presence of the cysteine residue in the binding domain. While we have not tested this hypothesis on MDMX, we have shown that a mutant of MDM2 protein (hMDM2(18–125)C77V) lacking the cysteine  examination of binding of MDMX immobilized to a surface by p53 peptide using surface plasmon resonance (SPR) and 2) examination of the thermal stability of MDMX.MDMX contains a single cysteine residue within the N-terminal p53 binding domain. Crystallographic studies have shown that the residue is buried within the protein (inaccessible to the solvent). Clearly, covalent modification would require at least partial unfolding of the protein. The initial hypothesis concerning reconciling the variation in alkylation with , Panels a and b; mimicking the conditions of the original assays), the p53 peptide does bind, but magnitude of the response is small (Panel a), the data quite noisy, and the resulting binding isotherm not well defined (Panel b). On the other hand, when the same protein on the same chip is reduced in situ (1 mM TCEP) and probed with p53 peptide, the response is robust (Panel c), the data clean, and the resulting binding isotherm well defined (Panel d). Of note is the fact that the measured Kd remains the same for both assays.The results of the SPR study are shown in d values suggest that the conformer responsible for binding p53 remains the same in both conditions while reversibility of the “inactive” state strongly suggests that the differences in behavior are due to changes in conformer populations.Thus, it appears that a substantial proportion of the protein in the non-reducing conditions is not functionally active. However, this is not a case of classic denaturation but rather a partial unfolding because the protein remains dis-aggregated on analytical chromatography (data not shown) and regains function with addition of reducing agents. The comparable K1 upon the conformational equilibria of MDMX were examined by a different technique – thermal stability as measured by hydrophobic dye binding 1 increases the temperature required to reach a phase transition, which would normally be interpreted as increasing stability. Our prior work has shown that similar covalent inhibitors of protein interactions often show slow on rates, relative to non-covalent inhibitors, and will show time dependencies in their behaviors. 1 (at apparent EC50 from the first experiment) reversed the stabilization of the protein caused by compound 1. When used alone, TCEP actually destabilized the protein at high concentrations while DTT had no apparent effect. This study strongly suggests that the binding of 1 to MDMX is reversible and that its effect is suppressed by reducing agents, whether or not they contain a nucleophilic thiol.Next, the effects of buffer condition changes and exposure to compound  binding Figure 51 and the cysteine residue is truly as depicted in 1 could form stable adducts with glutathione and with cysteine containing peptides as detected by LC/MS (S2). This was also true with other analogs , which is known to form mixed dithianes, the protein became unable to bind p53 peptide and the melting point was partially stabilized (S4). This raises the possibility that DTT, which is capable of forming such species might reverse the effects of compound 1 by inducing formation of a new protein adduct.Key issues arising from these studies are whether or not the interaction between compound 1 was allowed to interact with MDMX in the presence or absence of TCEP (a non-thiol reducing agent) and binding monitored by SPR. TCEP is neither expected to trap compound 1 nor form adducts with MDMX. When MDMX was immobilized to the chip and then treated with compound 1 in the absence of reducing agent, there was clear strong signal representing binding  and after the initial binding a decay of signal indicating that compound 1 binds reversibly. The off rate was relatively slow, requiring almost 5 minutes to return to baseline after the pulse of compound 1. If the same experiment was carried out in the presence of the non-nucleophilic reducing agent no binding was observed (Panel b). In control experiments where compound 1 was exposed to an excess of TCEP in the same buffers used for the MDMX protein experiments, no adduct formed between compound 1 and TCEP (data not shown). While not unexpected, this clearly indicates that compound 1 should be available for covalent reaction with the reduced cysteine. This study indicates that the binding of 1 to MDMX is reversible and requires access to a conformation that is suppressed by reducing conditions. This also explains why no covalent adduct is observed after treatment of MDMX with 1 when the protein is dialyzed prior to mass spectrometry – the dialysis time was sufficient to allow reversal of the reaction.In order to address these issues, compound 1. MDMX exists in a dynamic conformational equilibrium of at least two states that is biased both by the effects of compound 1 and reducing agents. In one state, which is favored by non-reducing conditions and concentrations of compound 1 above the solubility limit, MDMX is not competent to bind p53 and the side chain of cysteine 76 is exposed to the solvent and thus available for alkylation. Alkylation of Cys76 with 1 stabilizes this form, thus explaining the effect of compound 1 on p53 binding. A second state is favored in the presence of reducing agents and inhibitor concentrations below the solubility limit. In this state MDMX is able to bind p53 and the cysteine 76 side chain is buried inside the protein and thus resistant to alkylation. The fact that irreversible alkylation with DTNB also blocks p53 binding suggests that any alkylation of cysteine 76 will result in a conformational bias towards the conformation unable to bind p53. The mechanism by which reducing agents push the equilibrium towards the p53 binding competent conformation is unclear and may not simply be explained by forcing Cys76 into a reduced state.The studies described above suggest a consistent model of MDMX inhibition by compound 1 and reducing agents. Taken together these studies demonstrate that MDMX exists in conformers that are respectively either 1) relatively conformationally closed and competent to bind p53 peptide; or 2) relatively conformationally open and incompetent to bind p53 peptide. These conformational states appear to be in equilibrium with one another. Compound 1 appears to lock the protein into the latter state while reducing agents push it towards the former. These findings highlight the complex mechanism of compound 1, which does not appear to act as a simple competitive inhibitor but rather by affecting the conformation of its target protein. The study also emphasizes the role of the single cysteine residue in the p53 binding domain of MDMX which appears to be critical both for controlling the conformational equilibria of the protein and for the mechanism of action of compound 1. The originally reported data on reversibility of 1 were thus only a part of the full story uncovered here. This complex, multi-mode mechanism complicates the interpretation of experiments using 1 and limits its value as a lead compound for further development as a selective MDMX inhibitor.Thus, the MDMX protein exists in a complex conformational equilibrium that is biased both by the effects of compound E. coli. The bacterial cells were collected by centrifugation and lysed by sonication. Lysates were cleared by ultra centrifugation . For the GST-tagged version the supernatant was purified using a GSTrap Fast-Flow column . For the His-tagged version, the protein was purified using Talon nickel resin and the His-tag was subsequently removed by proteolytic cleavage. In both cases, the eluted protein was further purified by a Mono Q anion-exchange chromatography and gel filtration using S200 column. Peak fractions were combined and dialyzed into phosphate-buffered saline (pH 7.6) with 2 mM phenylmethylsulfonyl fluoride. Biotin-MDM4 was prepared using avi-tagged MDM4 (a.a. 23–111) and biotin labeled as recommended by the vendor (Genecopeia).A GST fusion protein of the p53-binding domain of human MDMX (a.a. 1–185) was cloned into a pGEX-4T1 plasmid. A second construct expressing a shorter hMDMX (a.a. 23–111) with a HIS-tag into a pGEX plasmid. Both constructs were expressed in BL21(DE3) The ITD measurements were performed with a RT-PCR instrument (Applied Biosystems 7900HT) equipped with a 384-well probe using SYBR detection (excitation 495/emission 537). The program used started at 45°C and the temperature increased at a rate of 1 degree per minute. The final concentrations were 0.125 mg/ml of GST-hMDMX (a.a. 1–185), varying concentrations of test compound (25 nM to 100 µM) and Sypro orange (Invitrogen) at 5-fold the protein concentration in a buffer consisting of 10 mM TRIS (pH 8.0) and 25 mM NaCl Solubility assay was carried out on Biomek FX lab automation workstation  using µSOL Evolution software . The detailed method is described as following. 10 µL of 10 mM compound stock (in DMSO) was added to 190 µL 1-propanol to make a reference stock plate. 5 µL from this reference stock plate was mixed with 70 µL 1-propanol and 75 µL SSB  to make the reference plate, and the UV spectrum (250 nm –500 nm) of the reference plate was read. 6 µL of 10 mM test compound stock was added to 594 µL SSB in a 96-well storage plate and mixed. The storage plate was sealed and incubated at room temperature for 18 hours. The suspension was then filtered through a 96-well filter plate . 75 µL filtrate was mixed with 75 µL 1-propanol to make the sample plate, and the UV spectrum of the sample plate was read. Calculation was carried out by µSOL Evolution software based on the AUC (area under curve) of UV spectrum of the sample plate and the reference plate. All compounds were tested in triplicates.50 values were determined by fitting observed mP values to a 4-parameter sigmoidal binding curve using PRISM 5.0 .All fluorescence polarization assays were performed in binding buffer  unless otherwise noted. For competition assays a master mix of 15 nM Texas red-labeled  P53 Peptide (sequence: GSGSSAETFSDLWKLLPEN) with 1 µM hMDMX  was prepared and 20 µl was added to black polystyrene 384 well plates (Corning #3573). DMSO stock solutions of test compounds were then added by pin transfer (V&P scientific) in nano-liter volumes . The assay mixture was incubated at room temperature for one hour and read on an EnVision multi-label plate reader using a 555 nm excitation filter, a 632 nm emission filter, and a Texas Red FP dichroic mirror. Binding inhibition curves and ICSamples were prepared in binding buffer  with a 50 µl final volume. Final concentration of test compounds (0 to 100 µM) and hMDMX constructs (1 or 20 µM) were varied as indicated in figure legends. Samples were incubated at room temperature for 1.5 hours after which they were stored overnight at 4°C before analysis by mass spectrometry. Samples containing 1 µM hMDMX constructs were concentrated to 20 µM using 3000 MWCO centrifugal protein concentrators  immediately prior to analysis. Prior to injection all samples were desalted using a reverse phase C8 Zip Tip (Millipore) and eluted in 50% acetonitrile, 2% formic acid. The samples were ionized by static nanospray using EconoTips (New Objective) on a Waters LCT Premier XE mass spectrometer using positive mode. The resultant charge envelope was deconvoluted using MaxEnt 1 algorithm of MassLynx V 4.0 sp 4 software. A mass error of 1Da for every 10,000 Da is permissible using this mass spectrometer.N-ethyl-N ´-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). For peptide experiments, streptavidin was attached at pH 4.5 in 10 mM sodium acetate to levels of ∼3000 RU per flow cell. For small molecule experiments, NeutrAvidin was attached at pH 5.0 in 10 mM sodium acetate to levels of ∼11000–12000 RU per flow cell. Any remaining reactive sites were blocked by reaction with ethanolamine. Biotin-MDM4 was captured on the chip by injection over the streptavidin or NeutrAvidin surface.Binding studies were performed at 25°C using a Biacore T100  surface plasmon resonance (SPR) instrument. Either streptavidin or NeutrAvidin (Thermo Scientific) was immobilized on a carboxymethyldextran-coated gold surface  by standard amine coupling methods. The carboxymethyl groups of dextran were activated with 1) was prepared in binding buffer  and injected at a single concentration of 100 µM at a flow rate of 100 µL/min. Data were processed, double-referenced and solvent corrected (where appropriate) using the software package Scrubber2 . Equilibrium dissociation constants (Kd) for peptide binding were determined by equilibrium affinity analysis with a 1∶1 binding model using Scrubber2.For peptide binding experiments, p53wt peptide was prepared as a 3-fold serial dilution in peptide binding buffer . The peptide was injected in triplicate at each concentration at a flow rate of 75 µL/min. In non-reducing conditions the peptide concentration range was 38 µM –469 nM, and in reducing conditions it was 19 µM –235 nM. For small molecule binding, compound SJ-172550 Click here for additional data file.Figure S2Formation of adducts by cysteine containing peptide and compound 1.(TIF)Click here for additional data file.Figure S3Inhibition of interaction of p53 and MDMX with DTNB.(TIF)Click here for additional data file.Figure S4Partial Stabilization of melting point of MDMX with DTNB.(TIF)Click here for additional data file.Figure S5Effects of glutathione upon potency of compound 1 and nutlin in blocking binding of p53 peptide by MDMX.(TIF)Click here for additional data file.Figure S6C77A mutation abolishes the ability of compound 1 to covalently label MDM2.(TIF)Click here for additional data file.Figure S7Formation of adducts by hMDMX(1–134) and compound 1.(TIF)Click here for additional data file.Figure S8Formation of adducts by hMDMX(1–111) and compound 1.(TIF)Click here for additional data file.Figure S9Formation of adducts by hMDM2(1–118) and compounds 1 and 3.(TIF)Click here for additional data file.Figure S10Formation of adducts by hMDM2(1–125) and compound 1.(TIF)Click here for additional data file.Figure S11Formation of adducts by β-mercaptoetanol and compound 3 evidenced by NMR.(TIF)Click here for additional data file.Figure S12Reduced sensitivity of C77A mutant of MDM2 to compound 3 but not nutlin in blocking the interaction with p53 peptide.(TIF)Click here for additional data file.Table S1Binding affinities of close and distant derivatives of 1 towards Mdm2 and Mdmx.(PDF)Click here for additional data file.