The molecular weight determined by gel filtration came out to be 43.6 KDa. The UV spectra of cystatin on interaction with donepezil suggested a conformational change in the protein. The fluorescence spectra of BC-donepezil composite show structural changes indicating 40 nm red shift with significant increase in fluorescence intensity of cystatin in the presence of donepezil representing an unfolding of cystatin on interaction, which is an indication of side effect of donepezil during the use of this drug.When drugs bind to a protein, the intramolecular structures can be altered, resulting in conformational change of the protein. Donepezil, an Acetyl Cholinesterase inhibitor (AChE), is commonly prescribed to patients with Alzheimer's disease (AD) to enhance cholinergic neurotransmission. It is the “first-line” agents in the treatment of Alzheimer's disease used to improve cognitive function in the disease. In the present study, a cysteine protease inhibitor (cystatin) has been isolated from buffalo brain using alkaline treatment, 40 to 60% ammonium sulphate fractionation and gel filtration chromatography on Sephadex G-75 with % yield of 64.13 and fold purification of 384.7. The purified inhibitor ) was eluted as a single papain inhibitory peak which migrated as single band on native PAGE; however, on SDS-PAGE with and without beta mercaptoethanol ( Brain is exposed to a variety of neuromodulating agents given as therapy. The effect of these agents apart from generation of desired activity must be assessed to understand the mechanism of action and side effects of the drug if any.Alzheimer is the most common cause of dementia; it is a primary degenerative disease of the brain. Onset is usually late in life with increasing impairment of memory, cognition, linguistic ability, and judgment. It is a progressive brain disorder that gradually destroys a person's ability to learn, reason, and carry out daily activities , 2. Acetylcholine is an important neurotransmitter associated with normal functioning of the brain. The greatly reduced concentration of acetylcholine in the cerebral cortex is a significant factor in AD , 4. PeopChE inhibitors prevent the hydrolysis of ACh to choline and acetate in the synaptic clefts and result in activating cholinergic transmission .Donepezil is a piperidine-class AChE inhibitor, rationally designed especially for AD , 6. DoneDonepezil is the first agent to be successfully developed specifically for the treatment of cognitive decline associated with Alzheimer's disease; it is marketed under the trade name of Aricept and works as an acetyl cholinesterase inhibitor . It has β-peptides clearing [ Recent researches have shown that cathepsins are involved the processing of certain neuropeptides in the central nervous system (CNS)  where cyclearing .  Cystatins constitute a powerful regulatory system for endogenous cysteine proteinases (cathepsins) which are often secreted or leaking from the lysosomes of dying or diseased cells . They ar Family 1: also called as stefins include members of low molecular weight proteins (11 kDa) which lack disulphide bonds and carbohydrate contents. This family includes cystatin A, B, stefins C, and stefins D. Family 2: known as cystatin family represented by the inhibitors of a bit larger molecular weight proteins (13 kDa) as compared to stefins and possesses disulphide bond towards the carboxyl terminal with no carbohydrates. They are found both in the cells and body fluids. Common example is cystatin C. Kininogens or family 3 cystatins are large precursor molecules of the vasoactive kinins. They are single chain glycoproteins, which serve a variety of biological functions such as kinin delivery, induction of endogenous blood coagulation cascade, and mediation of the acute phase response. They are found only in blood plasma .Cystatins tightly bind and inhibit the activity of cathepsins, if the activity of cathepsins is not regulated it will lead to chronic diseases . It has β peptides, donepezil when supplemented it binds to AChE and prevents acetylcholine breakdown thus increases the level of Acetyl choline which is required for normal functioning of the brain [In normal person, cystatins bind to cathepsins and prevent amyloid formation; however in AD, the level of cystatin goes down and the level of cathepsins and AChE enzyme increase which leads to decreases in acetylcholine and leads to formation Ahe brain . An earlhe brain .  The aim was to find out if supplementation of donepezil has any effect on the activity of cystatin  in the mammalian system. If the activity of these proteases is not regulated, it will lead to protease and antiprotease imbalance, a cause of several diseases .So we have investigated whether cystatin binding with donepezil has any role in the proper action of drug or leads to what kind of side effects as well as to gain knowledge about any conformational change in cystatin effecting its activity.Our study shows that donepezil unfolds cystatin which may not be able to bind to cathepsins; therefore an imbalance of protease - antiprotease occurs in the presence of donepezil leading to considerable side effect of the drug as cystatins play significant role in several diseases like arthritis cancer and cardiovascular diseases . TherefoPapain (99% purity) was obtained from Sigma Chemical Company . Donepezil (an Alzheimer drug) was purchased from Ranbaxy (India). The solutions were prepared in 50 mM phosphate buffer of pH 7.4. Salts were purchased from Merck (India). The protein concentration was determined spectrophotometrically. All other reagents were of analytical grade, and double distilled water was used throughout.Purification of Brain Cystatins. Buffalo brain whole mass (150 g) was brought fresh from slaughter house in an ice bucket. It was thoroughly washed with water, thin membrane and nerves were removed by forcep, and the whole brain tissue was homogenized in 50 mM sodium phosphate buffer (300 mL) of pH 7.5 containing 0.15 M NaCl, 3 mM EDTA, and 2% n-butanol. After centrifugation at 11000 rpm for 15 minutes at 40°C, residue was discarded and the supernatant was further processed. The procedure involved a combination of alkaline treatment (pH 11.0), ammonium sulphate fractionation, and gel filtration chromatography. Buffalo brain was homogenized and fractionated with ammonium sulfate between 40 and 60%; it was then dialyzed against 50 mM sodium phosphate buffer pH 7.4 containing 0.1 M NaCl. Elution profile showed two protein peaks one major and one minor called as peak-I and peak-II. Peak-I corresponding to high molecular weight Buffalo Brain Cystatin had significant inhibitory activity and protein content; however, peak-II with insignificant proteins concentration and low inhibitory activity was not taken into consideration for further studies. Peak-I named as BC was purified with fold purification of 384.72 and yield of 64.13%. Papain inhibitory fractions of peak-I were pooled, concentrated, and checked for purity. Five milliliter fractions were collected and assayed for protein and cystatin activity. Homogeneity of the preparation was investigated by 7.5% PAGE [.5% PAGE .Fluorescence Spectra of Brain Cystatin with Drug. Brain cystatin (BC) (1 μM) was incubated for 30 min with increasing concentrations of donepezil in 0.05 M sodium phosphate buffer pH 7.5 in a final reaction volume of 1 mL at room temperature. Drug solutions were prepared in the same buffer. Fluorescence measurements were carried out on a Shimadzu Spectrofluorometer model RF-5301PC  equipped with a 150 W Xenon lamp and a slit width of 10 nm at 298 K. The fluorescence was recorded in wavelength region 300–400 nm after exciting the protein at 280 nm. The slits were set at 10 nm for excitation and emission. The path length of the sample was 1 cm.UV Spectra of Cystatin in the Presence of Donepezil. The UV measurement of brain cystatin in the presence and absence of drug was made in the range of 200–300 nm, and the inhibitor (cystatin) concentration was fixed at 1 μM while the drug concentration was varied from 0.16 μM−1.6 μM. Absorption spectra were recorded on a double beam Shimadzu UV-vis spectrophotometer UV-1700 using a cuvette of 1 cm path length.Activity Measurement of Brain Cystatin in the Presence of Donepezil. The inhibitory activity of the purified inhibitor (BC) under native conditions was assessed by its ability to inhibit caseinolytic activity of papain by the method of Kunitz [μM) was incubated with increasing concentrations of donepezil at 25°C for 30 min before the activity was measured. Activity of untreated BC was taken as 100%.f Kunitz . The inh Alzheimer disease is a progressive brain disorder that gradually destroys a person's memory and ability to learn, reason, and make judgments. AChE is responsible for degradation of the neurotransmitter acetylcholine (ACh) in the synaptic cleft of neuromuscular junctions and of neuronal contacts in the central nervous system , 23. DonμM) was incubated with various concentrations of donepezil varying from 2 to 10 μM for 30 min. The fluorescence was recorded in the wavelength region of 300–400 nm after exciting the protein solution at 280 nm for total protein fluorescence. Donepezil caused unfolding of the cystatin as indicated by enhancement in fluorescence intensity accompanied by the red shift of 40 nm as compared to λmax of native cystatin (340 nm) while the drug (native) shows λmax at 370 nm. However at 1.6 μM, when it forms complex with cystatin there was shift in λmax of 10 nm with significant enhancement in fluorescence intensity ( Cystatin (1 ntensity .μM) was incubated with various concentrations of donepezil varying from 0.16 μM to 1.6 μM for 30 min. The fluorescence was recorded in the wavelength region 300–400 nm after exciting the protein solution at 280 nm for total protein fluorescence. The slits were set at 10 nm for excitation and emission. The path length of the sample was 1 cm in the final reaction volume of 1 mL in 0.05 M sodium phosphate buffer pH 7.5. Cystatin . Exposure of cystatin to increasing concentration of donepezil resulted in rapid decline of antiproteolytic activity; 85% decline in the activity was seen at 1.6 μM of donepezil with more than half of the inactivation of cystatin was taking place at concentration as low as 0.32 μM. A change in the inhibitory activity of cystatin with increasing concentration of donepezil is shown in . Effect μM) was treated with varying concentration of donepezil (0.16 μM–1.6 μM) for 30 min in the final reaction volume of 1 mL in 0.05 M sodium phosphate buffer pH 7.5. All data are expressed as mean ± S.E. for three different sets of experiments; statistical significance was conducted employing ONE WAY ANOVA. A probability level of 0.05 was selected showing that results are significant.Drug inducing changes in protein function leading to adverse side effects is the area of continual scientific investigation , 26. Even small structural differences in protein conformation can lead to drastic changes in functional parameters . AdditioAccumulation of drug molecules at certain sites in the body causes their localized high concentration leading to adverse drug reactions  and ligaIn the present work, structural and functional analysis of cystatin, a protein ubiquitously present in mammalian cells and tissues, was studied which showed a significant increase in fluorescence intensity due to unfolding of cystatin in the presence of donepezil . Such kiλmax indicating exposure of aromatic residues to the solvent caused by conformational changes in the protein [μM donepezil (μM donepezil (μM; cystatin the inhibitor retained only 15% of its antiproteolytic potential. Donepezil-cystatin complexation showed 40 nm red shift in  protein , 35. Absonepezil . suggestonepezil . Further Thus, the results indicate that the UV absorption and fluorescence emission changes in donepezil mediated interaction are due to conformational changes in cystatin mainly arising from interaction affecting the chromophoric groups of the protein which produce significant effect on the activity of cystatin. The study shows that in the presence of donepezil, cystatin gets unfolded which is a side effect of donepezil. The knowledge about the pharmacokinetics and pharmacodynamics of the drug-protein interaction continues to expand. The increased information available to clinicians might help in optimizing the use of these agents in the management of patients with Alzheimer's and other diseases. The clinical utility of measuring these parameters in daily practice awaits further research .


Objective(s): The role of the Apoptosis repressor with caspase recruitment domain (ARC) in apoptosis and in certain hypertrophic responses has been previously investigated, but its regulation of Endothelin-1 induced cardiac hypertrophy remains unknown. The present study discusses the inhibitory role of ARC against endothelin–induced hypertrophy. Results: In present study Endothelin treated cardiomyocytes were used as a hypertrophic model, that were subsequently treated with adenovirus ARC and its mutant at different multiplicity of infections. Casein-kinase-2 inhibitors were used to produce dephosphorylated ARC and to study its effect on hypertrophy. Hypertrophy was assessed by cell surface area measurement, Atrial-natriuretic-Factor mRNA analysis and total protein assay. Reactive oxygen species analysis was carried out using the dichlorofluorescin-diacetate (DCFH-DA) assay. Over expression of ARC significantly inhibits Endothelin–induced cardiomyocyte hypertrophy. The nonphosphorylated mutant ARC (T149 A) remained unable to control endothelin–induced hypertrophy, suggesting a vital role for ARC phosphorylation in regulation of its activity. Sensitization study has been carried out to check the role of endogenous ARC using casein-kinase inhibitors. Finally, the significant role of ARC in regulating reactive oxygen species -mediated control of endothelin induced hypertrophy has also been assessed.Conclusion: Conclusively, present study showed the vital and potential therapeutic interventional role of ARC in preventing endothelin-1–induced cardiomyocyte hypertrophy. The regulation of hypertrophic pathway by ARC relies on blunting the reactive oxygen species attack. This study further suggests a mediatory role of casein-kinase-2 in Endothelin–induced hypertrophy, mainly through its phosphorylation of ARC. Sustained cardiac hypertrophy is often accompanied by maladaptive cardiac remodeling, leading to heart failure . A fundaThe apoptosis repressor with caspase-recruitment domain (ARC) is abundantly expressed in the heart, which makes it a unique and central cardioprotective agent for the heart . Many stIn vitro studies in the neonatal rat have shown that ET 1–induced cardiac hypertrophy involves various hypertrophic signaling cascades, such as those involving protein kinase, Raf-1, and mitogen-activated protein kinases, which are mediated by the ET–type A (ETA) receptors -generating pathway is one among the complex signal-transduction pathways that can mediate hypertrophic signals. ROS can mediate the hypertrophic signals of TNF-α, PE, Ang II , and ET-Animal studies were performed in compliance with animal-welfare regulations of local authorities. ET-1, TBB, DRB, and Phalloidin tetramethylrhodamine isothiocyanate were purchased from Sigma . 2’, 7’-dichlorofluorescin diacetate (DCFH-DA), was purchased from Molecular Probes Inc.Construction of adenoviruses harboring ARC The adenoviruses harboring the wild-type rat ARC (AdARC) and an ARC mutant with T149 converted to the alanine residue (AdT149AββPreparations and transfection of ARC antisense oligonucleotides-ARC antisense oligonucleotides were synthesized to inhibit endogenous ARC expression. The sequences of phosphothioate-modified antisense oligonucleotides targeted to ARC were ARC antisense oligonucleotides (ARC-AS), 5’-TGGGCATGGAGGGTCATAGCT-3’; scrambled ARC antisense oligonucleotides (S-ARC-AS), 5’-GTAGGCTGAGGTCGATCGGTA-3’ and ARC sense oligonucleotides (ARC-S), 5’-AGCTATGACCCTCCATGCCCA-3’. The specificity of the oligonucleotides was confirmed by comparison with all other sequences in Genbank using Nucleotide BLAST. There was no homology to other known rat DNA sequences. Cells were transfected with the oligonucleotides by using lipofectin (Life Technology). Isolation and culture of cardiomyocytesNational Institutes of Health . Briefly, hearts were washed after dissection, minced in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid –buffered saline solution containing (in mM): NaCl, KCl, NaH2PO4, glucose, and HEPES in the ratio 130:3:1:4:20 (pH adjusted to 7.35 with NaOH). The tissues were then dispersed in a series of incubations at 37°C in HEPES-buffered saline solution containing 1.2 mg/ml pancreatin and 0.14 mg/ml collagenase (Worthington). After centrifugation, the cells were resuspended in Dulbecco’s modified Eagle’s medium/F-12 (GIBCO) containing 5% heat-inactivated horse serum, 0.1 mM ascorbate, insulin-transferring-sodium selenite media supplement, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.1 mM bromodeoxyuridine. The dissociated cells were preplated at 37°C for 1 hr. The cells were then diluted to 1 106 cells/ml and plated in different culture dishes coated with 10 µg/ml laminin, according to specific experimental requirements. After 24 hr, the medium was replaced by a serum-free medium.Cardiomyocytes were isolated from 1- to 2–day-old Wistar rats as previously described , 19. ExpAdenovirus infection and CK2 inhibitionCardiomyocytes were infected with adenoviruses as previously described . In expeCell surface–area measurementCell-surface areas of F-actin stained cells or unstained cells were measured after applying hypertrophic stimuli by computer-assisted planimetry. To determine the changes in cell size, the peripheries of cell images captured by a charge-coupled device camera  were traced and analyzed using NIH Image software. In each experiment, 100–200 cardiomyocytes were examined in 20–50 fields.(3H) leucine incorporation3H) leucine (1.0 µCi/ml). Thereafter, cells were washed 3 times with PBS, incubated with 5% trichloroacetic acid for 20 min at 4°C, and lysed with 0.5 M NaOH. Scintillation fluid was applied to the lysates, and the mixtures were counted in a liquid scintillation counter.Cardiomyocytes were infected with AdARC or Adβ-gal. 24 hr after infection they were treated with the hypertrophic stimuli for 48 hr in the presence of . Hybridization was performed in the same buffer and temperature for 30 hr with digoxigenin-labeled ANF cDNA probe. For chemiluminescent detection, the membrane was blocked for 30 min in 2.5% blocking reagent and then incubated for 30 min with anti-digoxigenin antibody conjugated with alkaline phosphatase. After two washes with 100 mM maleic acid buffer containing 0.3% Tween-20, CSPD substrate solution was added to the membrane and incubated for 10 min. The same membrane was stripped and re-probed with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control.Atrial natriuretic factor (ANF) expression was detected by Northern blotting as reported previously . BrieflyImmunoblottingImmunoblotting was performed as described . In brieIntracellular ROS analysisIntracellular ROS levels were analyzed using the ROS-sensitive dye, DCFH-DA, as described . DCFH-DA2O2; 30% w/v) was diluted in distilled water to a 20 mM stock solution and used at a final concentration of 200 µM as a positive control because of its known capacity to induce intracellular oxygen radical and hydrogen peroxide production in human cells. The fluorescence of 2',7'-dichlorofluorescein (DCF) was measured by flow cytometry and confocal fluorescence intensity–imaging microscope.Hydrogen peroxide  leucine incorporation method. This increase can be prevented by ARC overexpression  was expressed at a multiplicity of infection 100, whereas Ad-gal was considered the adenoviral control. Appropriate multiplicities of infection of adenoviruses were determined after numerous experiments with varying ranges. The cardiomyocyte hypertrophic model was set up by applying 0.1 µM ET-1 as described , 21. As pression . These rARC mutant, nonphosphorylated at 149 position, unable to inhibit ET 1–induced cardiomyocyte hypertrophy βInhibition of Endogenous ARC phosphorylation sensitizes cardiomyocytes to undergo ET 1–induced hypertrophyIn this phase of ARC sensitization experiments, endogenous ARC role in cardiomyocytes hypertrophy was analyzed by applying ARC antisense strand. Here very low dose of ET (5 nM) was applied that have no effect on cardiomyocytes hypertrophy as assessed by (3H) leucine incorporation method, but ARC antisense strand treatment inhibited endogenous ARC and sensitized cardiomyocytes to undergo hypertrophy . ARC antFor a better understanding of dependence of ARC on phosphorylation for its antihypertrophic effect, the authors carried out a study with the dephosphorylation of endogenous ARC. Because physiologically ARC is constitutively phosphorylated by CK2 , CK2 inhARC can control ET 1–induced cardiomyocyte hypertrophy by controlling intracellular ROSTo confirm the hypertrophic pathway followed by ET-1 and its subsequent inhibition by ARC, experiments to check the prevention of ET 1–induced increase in ROS levels by ARC were carried out. This study is also supported by the previous work by the authors of this study depicting regulation of catalase activity by ARC . These responsive elements provide the binding site to the gene products c-fos and c-jun responsible for hypertrophy and apoptotis . ROS, an important mediator of hypertrophy, plays an important role in neurohormone-induced hypertrophy because it has been shown to regulate the endogenous level of c-fos through the adapter protein 1 (AP-1) or Ras pathway under ET-1 stimulation . FurtherThe current studies indicate clearly that ARC can abrogate the ET 1–induced hypertrophy, whereas endogenous ET-1 can lead further to hypertrophy if inhibition of CK-2 occurred. In present study we have used varied concentrations of ET-1 under different scenarios. In order to induce hypertrophy ET 0.1 µM  and 2 waPrevious reports mentioned that ET-1 stimulation contributes to different cardiac disorders by activating the vascular endothelial growth factor (VEGF), Ap-1, Jun N-terminal kinase–, extracellular signal-regulated kinase–, and ROS-related pathways , 21. ET-Previous studies have reported the subcellular localization of different isoforms of caplain in the cytosol (μ and m) and in the mitochondria (I and II) . It was The current study shows that ARC can block the cascade of hypertrophic stimuli by blunting the ROS pathway. Furthermore, it can be hypothesized from the current study that there may be a direct inhibitory relationship between the (i) ROS-activated AP-1, cfos, VEGF, HIF-1α, and ARC-related control of HIF-2 in the mitochondria and (ii) the presentation of SOD and catalase for reinduction of CK-2 activity .in vivo research in the mouse model, based on the findings of current studies and presented hypothesis, can lead to novel therapeutic approaches for the treatment of maladaptive cardiac hypertrophy leading to cardiac failure. The antihypertrophic effect of ARC occurs through the scavenging of ROS generated because of neurohormone, ET-1 stimuli. Furthermore, the current study also shows the augmenting role of CK2, which is believed to be responsible for ARC phosphorylation at the endogenous level, in inhibiting ET1–induced hypertrophy. Future


Lactobacillus acidophilus (LA 5) in freeze-dried form was inoculated into a mixture of ice cream, frozen, and the number of viable organisms during frozen storage for a period of time was enumerated, using turbidity measurements with a spectrophotometer. An ice cream sample prepared without the probiotic culture was compared with the test sample for quality, by testing the basic quality parameters for ice cream. Results show a reduction in the over run of the probiotic ice cream compared to the nonprobiotic ice cream. Significantly high level (P < 0.05) of total solids (42%), proteins (16.5%), and titratable acidity (2.2%) was observed in the test sample compared to the nonprobiotic ice cream. Significantly low pH level in the probiotic sample may be due to the lactic acid produced by the probiotic culture. No significant difference (P > 0.05) in the fat content in the two types of ice cream was observed. A significantly low level (P < 0.05) of melting in the probiotic one may have resulted from less over run, than the nonprobiotic sample. Rapid reduction in the viable cells during frozen storage occurred at −18°C and gradual adaptation occurred over the first 4 weeks. At the 10th week, 1.0 × 107 numbers of viable organisms were present in 1 g of the probiotic ice cream. Results show the presence of a sufficient number of viable organisms in the product for the 10-week period, which would be beneficial to consumers.A study was performed to apply a probiotic strain into fermented ice cream mix with suitable fruit bases to develop a value-added product with a substantial level of viable organisms for a sufficient shelf life. Pure direct vat strain culture of  Lactobacillus acidophilus) is commonly used around the world in food products. Various criteria for the selection of probiotic strains were proposed based on safety, efficacy, and technological production . Probiotics, when ingested beneficially, affect the host by replenishing the depleted gut microflora, which may have occurred due to the use of antibiotics, illness and stress, travel or lifestyle changes, and also by improving the properties of the indigenous microflora of the host. Microbial strains for probiotic use must be representative of microorganisms that are generally recognized as safe microbes. The strain used in this study  was obtained from J.L. Morison, Sons & Jones (Ceylon) Ltd, Colombo, in freeze-dried form and the samples were kept at −18°C. Fresh milk was obtained from National Livestock Development Board (NLDB) farms, Sri Lanka, for the preparation of the ice cream mix.Pure direct vat strain culture of An ice cream mix consisting of 37.4% of total solids (TS), 10% of fat, 11% of milk solid nonfat, 16% of sugar, 0.4% of stabilizer–emulsifier mix, and 63.6% of water was formulated. The required quantities of the ingredients for 900 mL of ice cream mix  were weighed, mixed, and heated for 50°C and the mixture was homogenized using a laboratory-scale blender for 5 min. The homogenized mixture was pasteurized at 90°C for 15 min and 50% of the prepared ice cream mix was kept at 4°C and aged for 18 h.Lactobacillus acidophilus culture (0.1 g) was transferred into unaged ice cream mix and mixed thoroughly under sterile conditions. The mixture was incubated at 43°C for 3 h and refrigerated at 6°C to prevent further fermentation.Fresh wood apples were cleaned, cracked, and seeds were removed. They were mixed with a 1:1 ratio of potable water and liquidized. The mixture was pasteurized at 63°C for 30 min and refrigerated at 6°C.Ice cream and yogurt mix (50% each), together with 100 mL of wood apple puree making the total volume of 1 L, was transferred into a batch freezer and air was incorporated until the volume of the total mix reached nearly the double of its initial volume. The frozen mixture was transferred into sterile containers and refrigerated at −18°C to harden the product.L. acidophilus.An ice cream was prepared as the control with 900 mL of ice cream mix and 100 mL of fruit puree without the addition of X1) and after whipping (X2) (% over run = X2 − X1/X1 × 100). TS, fat, and protein contents were analyzed using Association of Official Analytical Chemistry (AOAC) (Melting rate was determined as the average time taken to drip a scoopful of ice cream (melting rate = 1/time [min]). Over run was calculated as the weight of the ice cream mix before whipping (y (AOAC)   . The dilutions were selected in the absorbance range 0.2–0.7 and the particular samples were cultured in nutrient agar using the pour plate method.−2–10−6) was added into sterile petri dishes and 15 mL of nutrient agar was poured into each dish. The petri dishes were mixed well and incubated at 37°C for 24 h and the colonies were counted between 15 and 300 and the number of colony-forming units (CFU) in 1 mL of dilution was determined (CFU = no. of colonies × dilution factor).The number of organisms was measured by adding molten agar (pour plate method). One microliter of each dilution (10A graph was plotted between the number of CFU in 1 mL dilution versus absorbance. Samples were taken from the prepared ice cream within different time periods (up to 10 weeks) while in frozen storage (−18°C) and the absorbance of prepared dilutions was measured (selected the dilutions in the 0.2–0.7 range). Using the standard graph, the number of organisms in the sample was determined.Total plate count of microbes using nutrient agar was carried out in probiotic and nonprobiotic ice creams when the products were frozen after production using the bacterial enumeration method (pour plate technique).Samples were triplicated and results were compared by analysis of variance (ANOVA) using MINITAB ver 14 .P < 0.05) than in the nonprobiotic ice cream and the significantly high level of titratable acidity and the low pH level in the test sample are probably due to the lactic acid produced by the probiotic culture. There is no significant difference (P > 0.05) in the fat content of the two types of ice creams and a comparatively low level of melting was observed in the probiotic than in the nonprobiotic sample. The low level of melting may have resulted from the foam stability arising due to the low pH, coupled with the higher level of TS in the probiotic product. The amount of air also determines the melting rate and it is evident that low over run compared to nonprobiotic product may have been associated with the low melting rate of the probiotic ice cream.Results of the basic ice cream tests show a reduction in the over run of the probiotic-added ice cream compared to the nonprobiotic ice cream Table . TS and 12 and 1 × 102. From this observation, we can assume that the excess number of 1 × 1010 organisms in the test sample is due to the probiotic culture. Shelf life studies after the first week of frozen storage indicate a twice than 10-fold reduction in the initial number of organisms . Therefore, the test sample can cover this requirement for more than 10 weeks. More viable organisms can ensure better survival of probiotics in the gut and be beneficial consumers who use the product on a daily basis over a period of time. Depending on the individual, the required quantity and time required for successful colonization in the gut vary. It is suggested to feed the product into selected groups of individuals for a period of time followed by enumeration of organisms in the stool samples for a more conclusive study.The rapid reduction in viable cells during frozen storage may be due to the inability of the organisms to adapt to the rather low level of temperature at once (−18°C). The adaptability of the remaining organisms to the existing environment is shown in the first 2 weeks. A 10-fold reduction in viable organisms was observed between 6 and 10 weeks of frozen storage. A gradual decrease in the number of organisms can be seen over the 10-week period and at the end of the 10th week, 1.0 × 10Probiotic ice cream shows a reduction in the over run, high level of TS and protein contents than the nonprobiotic ice cream. Increased titratable acidity and reduced pH together with low over run resulted in a low melting rate in the LA 5 direct vat strain culture-added ice cream. The number of organisms was reduced significantly in the initial stage of frozen storage and gradual reduction occurred over the tested period of time while a considerable number of organisms survived and provided beneficial effects.


Amanita phalloides. Here we show that antamanide inhibits the mitochondrial permeability transition pore, a central effector of cell death induction, by targeting the pore regulator cyclophilin D. Indeed, (i) permeability transition pore inhibition by antamanide is not additive with the cyclophilin D-binding drug cyclosporin A, (ii) the inhibitory action of antamanide on the pore requires phosphate, as previously shown for cyclosporin A; (iii) antamanide is ineffective in mitochondria or cells derived from cyclophilin D null animals, and (iv) abolishes CyP-D peptidyl-prolyl cis-trans isomerase activity. Permeability transition pore inhibition by antamanide needs two critical residues in the peptide ring, Phe6 and Phe9, and is additive with ubiquinone 0, which acts on the pore in a cyclophilin D-independent fashion. Antamanide also abrogates mitochondrial depolarization and the ensuing cell death caused by two well-characterized pore inducers, clotrimazole and a hexokinase II N-terminal peptide. Our findings have implications for the comprehension of cyclophilin D activity on the permeability transition pore and for the development of novel pore-targeting drugs exploitable as cell death inhibitors.Antamanide is a cyclic decapeptide derived from the fungus  Amanita phalloidesin vitroAntamanide (AA) is a monocyclic, homodetic decapeptide isolated from the poisonous mushroom cis-trans isomerase activity CyP-A is a component of the CyP protein family, whose members display peptidyl-prolyl Here we demonstrate that, similar to CsA, AA targets CyP-D leading to PTP inhibition and to cell protection from insults that cause pore opening. AA could be exploited as a lead compound for a new class of PTP-inhibiting drugs.2+ retention capacity (CRC) assays on isolated mouse liver mitochondria (MLM). Notably, when mitochondria were incubated in a phosphate-containing medium, AA inhibited pore opening, similar to the PTP inhibitors CsA or Ubiquinone 0  . To evale 0 Ub0; . PTP inhe 0 Ub0; . We had e 0 Ub0; . To disse 0 Ub0; . We foune 0 Ub0; .−/−Ppif (CyP-D null) mouse fibroblasts i.e. by measuring mitochondrial Ca2+ retention capacity on digitonized cells  detachment from mitochondria with the drug clotrimazole (CTM) or with a HKII-displacing peptide TAT-HK; . We founThe identification of PTP targeting drugs is a highly desirable result, as the PTP is involved in a wide range of diseases. By the use of CsA or of CyP-D knock-out animals it was established that dysregulated PTP induction is implicated in various forms of brain damage CyP-D is a promising pharmacological target. It is part of the CyP protein family, composed by ubiquitous and extremely conserved proteins located in different subcellular compartments To the aim of selecting these more efficacious drugs, a fine comprehension of the interaction with their molecular targets is needed. The CsA-binding pocket of CyPs is formed by an extended groove where the isomerase active site is located. CsA half-inserts in the cavity, and a high binding affinity to CyPs requires the sum of multiple interactions: six CsA amino acids (residues 1–2 and 8–11) establish hydrogen bonds and hydrophobic contacts with 15 CyP amino acids. Residues 3–7 of CsA are exposed to the outside of the CyP-CsA complex and are prone to interact with other molecules i.e. the isomerization to the cis form of the peptidyl bonds preceding each proline cis-trans isomerase activity of CyP-D. This observation strongly suggests that the enzymatic inhibition of CyP-D is the molecular mechanism responsible for PTP inhibition. Thus, AA could have diverse biological effects, as CyPs modulate a variety of cell processes, including gene transcription, proliferation, survival, chemotaxis and motility, targeting molecules as diverse as the transcription factor NF-AT, MAP kinase upstream regulators It must be highlighted that the biological activities of AA are possibly wider than the presently reported PTP inhibition. Indeed, both AA and CsA inhibit the PTP (present results), block cell uptake of the phallotoxin phalloidin ; Calcium Green-5N and tetramethylrhodamine methyl ester (TMRM) were from Molecular Probes ; dinitrophenol was from Merck ; digitonin was from Calbiochem ; all other chemicals were from Sigma . Diaphragm adult fibroblasts were obtained by SV40 immortalization of primary cells from wild-type and ab initio a C-terminal tert-butyloxycarbonyl-protected hydrazide moiety), 5–6 and 7–10. These fragments were subsequently assembled by the azide method following the Rudinger procedure obtaining the linear 5-4 sequence of AA. The same method was used for the cyclization reaction exploiting the possibility to separate the activation from the condensation which was carried out at concentration not exceeding 10−3 M in the presence of an inorganic base. Crude peptides were purified by elution on a Sephadex LH-20  column equilibrated and eluted with methanol-water (8∶2 v/v). Molecular masses of the peptides were confirmed by ESI-MS on a Mariner  mass spectrometer. The purity of the peptides, assessed by analytical reverse phase HPLC, was higher than 95%. The amino acid compositions of the peptide acid hydrolysates  were determined with a 3A30  amino acid analyzer. Peptides MIASHLLAYFFTELNbA-GYGRKKRRQRRRG (TAT-HK) and GYGRKKRRQRRRG-bA-EEEAKNAAAKLAVEILNKEKK (TAT-Ctr) were synthesized as described AA and four derivatives, Tyr9-AA, Gly9-AA, Gly6-AA, and octa-Gly9-AA were synthesized as reported elsewhere g, 10 min), and mitochondria were then spinned down at a higher speed . All procedures were carried out at 0–4°C.Mitochondria were isolated either from livers of C57BL/6 mice through sequential centrifugations, or from cells, as described 2+ retention capacity (CRC) assay was used to assess PTP opening following trains of Ca2+ pulses and measured fluorimetrically at 25°C in the presence of the Ca2+ indicator Calcium Green-5N . Experiments were performed either on isolated mitochondria or on whole cells c, and Calcium Green-5N, which does not permeate mitochondria. Cells or mitochondria were then exposed to Ca2+ spikes, and fluorescence drops were used to assess mitochondrial Ca2+ uptake. PTP opening was detected as a fluorescence increase. Calcium Green-5N fluorescence was measured either with a fluorescence spectrometer LS50B  or with a Fluoroskan Ascent FL fluorimeter .The CaCell extracts were prepared at 4°C in 140 mM NaCl, 20 mM Tris·HCl (pH 7.4), 5 mM EDTA, 10% glycerol, and 1% Triton X-100 in the presence of phosphatase and protease inhibitors (Sigma). Samples were then denatured, separated in reducing conditions on SDS-polyacrylamide gels and transferred onto Hybond-C Extra membranes . Primary antibodies were incubated 16 hours at 4°C, and horseradish peroxidase-conjugated secondary antibodies were added for 1 hour. Proteins were visualized by enhanced chemiluminescence .Mitochondrial oxygen consumption was measured polarographically at 25°C with a Clark oxygen electrode . Mitochondria  were incubated in a solution of 130 mM KCl, 10 mM Tris-Mops, 1 mM Pi-Tris, 20 mM EGTA-Tris and 2 µM rotenone/5 mM succinate to assay basal respiration (state 4). ADP (200 µM) was subsequently added to measure state 3 respiration, followed by the uncoupling agent dinitrophenol (100 µM) to assess the maximal respiration rate.2 and incubated at 37°C in either TMRM (10 nM) to detect mitochondrial depolarization (reduced TMRM staining), or in FITC-conjugated Annexin-V and propidium iodide , to detect phosphatidylserine exposure on the cell surface (increased FITC-conjugated Annexin-V staining) and loss of plasma membrane integrity (PI permeability and staining). When TMRM was used to detect mitochondrial membrane potential, in each experiment one sample was treated with the protonophore FCCP (4 µM) as a positive control . Moreover, as TMRM can be pumped out of cells by multidrug resistance systems, and CsA is a multidrug resistance inhibitor, the CsA analogue cyclosporin H, which is inactive on the PTP, was used to block multidrug resistance in the absence of CsA Flow cytometry recordings were performed as described cis-trans-Pro-Phe-p-nitroanilide by chymotrypsin, as previously reported trans form of the peptide, which is revealed as a rise in absorbance at 410 nM. The concentration of of the cis form is maximized by using a peptide stock dissolved in trifluoroethanol containing 470 mm LiCl, and its hydrolysis is limited by the rate of cis-trans isomerization.The PPIase activity was assessed on recombinant CyP-D . The CyP-D cDNA was cloned in a pcDNA3 vector  with a FLAG tag added at its 3′ end Figure S1Effect of phalloidin (Ph) on mitochondria and cells. A, ratio between the CRC detected in the presence (CRC) and absence (CRC0) of Ph (50 µg/ml) in MLM. The effects of CsA (0.8 µM) and AA (8 µM) are reported as positive controls. Results are mean±SD of 3 experiments. B, cytofluorimetric analysis of mitochondrial membrane potential after a 1 hour incubation with Ph (50 µg/ml). Treatment with the proton uncoupler FCCP (4 µM) is shown as a positive control of mitochondrial depolarization. Graphs report a representative experiments, where HeLa cells with polarized and depolarized mitochondria are indicated in red and grey, respectively. D is the percentage of cells with depolarized mitochondria.(TIF)Click here for additional data file.


Recently we described the reversible formation of highly phosphorylated tau protein in hibernating European ground squirrels. Hence, mammalian hibernation represents a model system very well suited to study molecular mechanisms of both tau phosphorylation and dephosphorylation under in vivo physiological conditions. Here, we analysed the extent and kinetics of hibernation-state dependent tau phosphorylation in various brain regions of three species of hibernating mammals: arctic ground squirrels, Syrian hamsters and black bears. Overall, tau protein was highly phosphorylated in torpor states and phosphorylation levels decreased after arousal in all species. Differences between brain regions, hibernation-states and phosphosites were observed with respect to degree and kinetics of tau phosphorylation. Furthermore, we tested the phosphate net turnover of tau protein to analyse potential alterations in kinase and/or phosphatase activities during hibernation. Our results demonstrate that the hibernation-state dependent phosphorylation of tau protein is specifically regulated but involves, in addition, passive, temperature driven regulatory mechanisms. By determining the activity-state profile for key enzymes of tau phosphorylation we could identify kinases potentially involved in the differentially regulated, reversible tau phosphorylation that occurs during hibernation. We show that in black bears hibernation is associated with conformational changes of highly phosphorylated tau protein that are typically related to neuropathological alterations. The particular hibernation characteristics of black bears with a continuous torpor period and an only slightly decreased body temperature, therefore, potentially reflects the limitations of this adaptive reaction pattern and, thus, might indicate a transitional state of a physiological process.Abnormal phosphorylation and aggregation of tau protein are hallmarks of a variety of neurological disorders, including Alzheimer's disease (AD). Increased tau phosphorylation is assumed to represent an early event in pathogenesis and a pivotal aspect for aggregation and formation of neurofibrillary tangles. However, the regulation of tau phosphorylation  Tau is an axonally located, microtubule-associated protein that is encoded by a single gene and predominantly expressed in neurons in vitro conditions in vivo models of PHF-like tau phosphorylation so far impedes a proof of this concept.A variety of neurodegenerative disorders is characterised by the formation of intracellular deposits of phosphorylated tau protein aggregated into paired helical filaments (PHF) We have demonstrated a PHF-like phosphorylation of tau in hibernating mammals Therefore, mammalian hibernation represents a model system very well suited to analyse conditions and mechanisms physiologically associated with increased tau phosphorylation and altered synaptic connectivity. In addition, the hibernation model helps to identify potential differences between tau hyperphosphorylation in torpor and AD, thereby contributing to our understanding of the significance of tau phosphorylation for neurodegeneration.Spermophilus parryii), Syrian hamsters (Mesocricetus auratus) and black bears (Ursus americanus). These species were selected since they differ with respect to their hibernation characteristics as well as their degree of body temperature change during torpor. With a body temperature of about 0°C arctic ground squirrels show the most extreme reduction followed by the Syrian hamsters where body temperature is lowered to about 5°C during hibernation. Black bears, however, show only a slight temperature change during hibernation with a body temperature of no lower than 30°C. In arctic ground squirrels and Syrian hamsters hibernation is characterised by regular torpor intervals interrupted by brief arousal episodes, where metabolic rate and body temperature of animals returns to normal levels. On the contrary, black bears do not show such regular arousal episodes.In the present study we analysed the reversible tau phosphorylation in arctic ground squirrels , cyclin dependent kinase 5 (cdk5), stress-activated protein kinase/Jun-amino-terminal kinase (SAPK/JNK) and mitogen activated protein kinase/extracellular regulated protein kinase (MAPK/ERK) during hibernation.The three species were compared using a panel of phosphorylation-dependent tau antibodies that was applied to different brain regions. To analyse whether hibernation-state related tau phosphorylation is a specifically regulated process or passively driven by a brain temperature-dependent shift of kinase- and phosphatase activities, we assessed the phosphate net turnover of tau protein in different hibernation-states. Moreover, to elucidate the molecular mechanisms regulating tau phosphorylation The hibernation-related tau phosphorylation was analysed immunohistochemically and by Western blot using different phosphosite-specific tau antibodies. A summary of the investigated tau phosphorylation sites and the applied antibodies is shown in Generally, tau protein is highly phosphorylated during torpor states and phosphorylation levels decrease after arousal as demonstrated in Antibodies that detect specific phosphorylation sites on tau protein were used to characterise the regional tau phosphorylation pattern during the hibernation cycle by Western blot. In total, seven phosphosites see  were anaThe analysis of results from arctic ground squirrels  and SyriThe tau phosphosite T231/S235 was the only site showing reversible phosphorylation in all brain regions analysed. Interestingly, for this site we also observed aberrant phosphorylation kinetics. In general, tau phosphorylation did not further increase in late torpor. However, phospho levels at T231/S235 were significantly elevated in late torpor compared to early torpor. This increase was detected in both arctic ground squirrels and Syrian hamsters indicating a conserved characteristic of this particular site.In black bears sampled during hibernation all seven of the investigated phosphosites showed an increased phosphorylation level compared to summer active animals, both in frontal cortex and hippocampus , Table 2We applied the Alz-50 antibody that specifically detects a particular tau conformation, typically for PHF-tau Tau protein expression was analysed by Western blot using a phospho-independent tau antibody. The results are listed in Regarding tau mRNA isoform expression we focused on the analysis of a potentially altered splicing of exon 10 that encodes for an additional microtubule binding repeat and thereby alters the binding capacity of tau. The analysis was performed in arctic ground squirrels  whereas prior to the isoform expression analysis Tau gene transcripts of were cloned and sequenced (GenBank accession number: FJ609677). Based on the determined sequence, species-specific primers were designed binding in exon 7 and exon 11 respectively. The PCR revealed two distinct PCR products with a size of about 390 bp and 290 bp corresponding to the predicted PCR product size depending on the presence or absence of exon 10 thereby demonstrating the expression of both 3-repeat and 4-repeat tau isoforms in adult arctic ground squirrels . To deteThe formation of phospho-proteins was determined in neocortical brain homogenates of arctic ground squirrels . There were no differences regarding the levels of phospho-proteins during hibernation .To determine whether tau phosphorylation during hibernation is specifically regulated or a passive process driven by temperature-dependent alterations of kinase- and phosphatase activities, we analysed the net turnover of phosphate in tau protein at different incubation temperatures. This assay is designed to determine both phosphorylation and dephosphorylation of tau by assessing the incorporation of radioactively labelled phosphate residues into the tau protein. Therefore, the assay reflects the result of kinase and phosphatase activities in the analysed brain extracts. The result of enzyme activities was assessed in cortical brain samples of arctic ground squirrels .First, we analysed the phosphate net turnover at various temperatures . ReactioTau protein phosphate net turnover was significantly altered  in both torpid and aroused animals as compared to euthermic animals. Most interestingly, there was no general shift towards increased phosphorylation in torpor and arousal. We rather determined a temperature dependent variation in tau phosphorylation in these hibernation states. The most intense phosphate intake was observed at 30°C in torpid animals resulting in an almost doubled phospho-tau degree.in vitro. Brain samples were taken from euthermic, torpid and aroused animals and subjected to a decreasing and an increasing temperature gradient. As shown in To further analyse this temperature dependency, we measured rate of tau phosphate net turnover at a series of temperatures that simulated the entry into torpor and arousing We determined brain tissue activity of the major tau kinases glycogen synthase kinase 3 beta (GSK3-beta) cyclin dependent kinase 5 (cdk5), stress-activated protein kinase/Jun-amino-terminal kinase (SAPK/JNK), and mitogen activated protein kinases/extracellular regulated protein kinase (MAPK/ERK). Activity of these kinases is regulated by phosphorylation, which increases enzymatic rates, except for GSK3-beta, where phosphorylation of residue S9 is inhibitory Phosphorylation of GSK3-beta and cdk5 was significantly increased in torpid animals, indicating inhibition of GSK3-beta and activation of cdk5 activity during torpor . Phosphoin vivo.In ground squirrels, the microtubule-associated brain protein tau enters a hyperphosphorylated state during hibernation that is fully reversed when animals periodically arouse to normal levels of metabolism and brain temperature Hibernation is a behavioural and physiological adaptation in mammals in which the need for energy is profoundly decreased during conditions of current or anticipated famine. Hibernating animals enter a state of reduced metabolism and decreased body temperature called torpor that can continue for several days to several weeks in small mammals and several months in large mammals. Hibernation is found in a variety of species in several different orders Mammalian hibernation is thought to be based on similar physiological mechanisms Spermophilus parryii), Syrian hamsters (Mesocricetus auratus) and black bears (Ursus americanus). These species were selected to determine whether tau phosphorylation is a general, hibernation-related phenomenon and whether species that hibernate with different body temperatures and patterns show the same patterns of phosphorylation change.Here we analysed the hibernation-state dependent tau phosphorylation in arctic ground squirrels  cyclin dependent kinase 5 (cdk5), stress-activated protein kinase/Jun-amino-terminal kinase (SAPK/JNK) and mitogen activated protein kinases/extracellular regulated protein kinase (MAPK/ERK) in arctic ground squirrels and Syrian hamsters. Interestingly, the results revealed a differentially regulated enzyme activity pattern. Generally, GSK3-beta is supposed to be the primary candidate kinase responsible for tau hyperphosphorylation whereas the other kinases are assumed to modulate tau phosphorylation The phosphorylation of cdk5 at S159, in contrast, indicates an increased activity in the state of torpor Spermophilus richardsonii) no hibernation-related change in JNK activity was found The decreased phosphorylation of SAPK/JNK indicates an inhibited activity during torpor. This finding is consistent with results showing decreased SAPK/JNK activity in torpid arctic ground squirrels The MAP-kinases ERK1 (p44) and ERK2 (p42) showed a differentially regulated activity pattern. ERK1 phosphorylation was increased while ERK2 was less phosphorylated in torpid animals. These results are contrary to findings reporting on a decrease of both ERK1 and ERK2 activity during torpor in arctic ground squirrels To summarise, we found cdk5 and ERK1 positively yet GSK3-beta, SAPK/JNK and ERK2 negatively regulated in torpid animals. The determined kinase activity-state pattern was analogous in both analysed species indicating equivalent regulatory mechanisms. Based on our findings cdk5 and ERK1 may act as kinases that actively phosphorylate tau under physiological conditions. Consequently, since hibernation is not associated with pathological consequences, activation of GSK3-beta and SAPK/JNK as demonstrated in Alzheimer's disease may indicate pathophysiological mechanisms.Unravelling the regulation of hypometabolic states such as hibernation are of great significance for the understanding of cellular and molecular aspects of neurodegenerative disorders where hypometabolic states of a “vita minima” precede cell death. As shown by functional brain imaging, these hypometabolic states occur very early during the course of AD, even in presymptomatic stages For that reason, physiological adaptations that are observed at the hypometabolic state in hibernation are potentially analogous to neuronal reactions to a hypometabolism in very early stages of AD.Both, the increased tau phosphorylation and the reduced expression of the four-repeat isoforms result in a decreased microtubule binding capacity of tau protein. This coincidence strongly suggests that this particular condition is one prerequisite for neurons to endure the state of torpor. In this regard the biological relevance of an increased phosphorylation of tau protein in preclinical stages of AD might be reconsidered. It is very likely that particular types of neurons respond to a hypometabolic state with an elevated phosphorylation of tau protein. Hence, this reaction represents a physiological mechanism of the cell and not a primary pathological event In vitro studies could demonstrate that the aggregation of tau is inhibited at low temperatures In the neocortex of hibernating black bears we found a conformational change of tau protein. This particular interspecies difference might be the result of the considerably elevated body temperature in torpor of bears. A PHF-like phosphorylation and altered isoform expression of tau protein occurs during torpor in three species of hibernators that differ greatly in body size and in the minimum brain temperatures and metabolic rates they achieve. This phosphorylation is fully reversed when animals return to normal levels of temperature and metabolism whether it is regularly during arousal intervals in small hibernators or seasonally in large hibernators. These findings indicate that a reduced binding capacity of tau may be a precondition to endure the hypometabolic state and reduced tissue temperatures during hibernation. Moreover, the interspecies homogeneity of this reaction pattern suggests that this regulation is subject to a basal physiological mechanism of mammals. Increased neuronal tau phosphorylation in early stages of AD, therefore, may be potentially considered as physiological reaction to a reduced brain metabolic rate. However, as the result of the slow and progressive pathological process in AD, hyperphosphorylated tau protein aggregates to neurofibrillary tangles most likely leading to the degeneration of affected neurons.PHF-like tau phosphorylation in hibernation is paralleled by an aberrant synaptic plasticity and a loss of memory. Due to the co-occurrence of these elementary attributes of AD hibernating mammals represent a unique and useful model organism to study the relevance and coherencies of particular characteristics of AD pathology. This is of utmost importance for the development of potential strategies for a medical treatment and therapy of this disease.Only animals in good health were used.Spermophilus parryii): Animal protocols were approved by the University of Alaska Fairbanks Institutional Animal Care and Use Committee .Arctic ground squirrels (Mesocricetus auratus): All experimental procedures on animals were carried out in accordance with the European Council Directive of November 24th 1986 (86/609/EEC) and had been approved by the local authorities .Syrian hamsters (Ursus americanus): Animal protocols were approved by the University of Alaska Fairbanks Institutional Animal Care and Use Committee  and USAMRMC Animal Care and Use Review Office .American Black bears  were implanted into the peritoneal cavity of each animal (body mass range 650–950 g). Before implant, transmitters were sealed in heat shrink tubing and triple coated in Elvax  creating a package weight of 17–20 g, calibrated to the nearest 0.1°C at 0 and 20°C against a precision mercury glass thermometer, and gas sterilized Arctic ground squirrels (n = 31) were trapped during August in the Alaska Range or on the North Slope of Alaska near Toolik Lake and transferred to University of Alaska Fairbanks. The animals were housed individually and provided with Mazuri Rodent Chow sunflower seeds, carrots, and apple slices and provided with water In this study, animals were compared at five stages of their annual cycle. Euthermic animals  that had ended hibernation and been continuously at high body temperatures for 1–2 months were sampled in May and June after having gone through their reproductive phase and entered spring molt. Animals in hibernation were sampled in mid winter when 3–5 days within a torpor bout  and after 10–12 days in a torpor bout . Animals that naturally rewarmed from prolonged torpor during an interbout arousal were sampled 2–3 h  and 10–12 h  after core body temperature increased above 30°C as indicated by the radiotransmitter. For sampling, summer active and naturally aroused animals were anesthetized with isoflurane, euthanized with sodium pentobarbital and decapitated. Torpid animals were euthanized by decapitation after verification of core body temperatures <7°C. Brains were removed from the skull, rapidly dissected, frozen in liquid nitrogen, and stored at −80°C.2 ventilation and decapitated. Subsequently, the brain was removed, dissected, immediately frozen in liquid nitrogen and stored at −80°C.Syrian hamsters purchased from Harlan Winkelmann GmbH  were bred and housed at the Medizinisch-Experimentelles Zentrum of the Medical Faculty of the University of Leipzig. In total, 41 animals were included in the study and subjected to hibernation conditions st-27th (n = 5). These animals were without food since October 27th and had been offered a diminishing food ration for two weeks prior. For monitoring of physiological conditions, bears used in hibernation experiments were surgically implanted in late November with telemetry transmitters for core body temperature  and transmitters for ECG and EMG monitoring  which transmitted both body core temperature and biopotentials. All transmitters were implanted intra-peritoneally. Beginning in late November, bears were housed in individual outdoor enclosures on the University of Alaska Fairbanks campus that had dens and straw material for nests. Dens were double walled boxes made from 2.5 cm thick plywood or high-density polyethylene with inside dimensions of approximately 0.8×1.0×1.0 m with 5 cm of Styrofoam between walls. Each nest box had a 46×46 cm door opening that was fitted with a double-layered breakaway door sealed by weather stripping and kept in place by metal clamps. The dens were instrumented with antennas and a thermocouple to monitor temperature. Air was continuously drawn from an exit hole at back of the den and oxygen consumption monitored as described previously 2 powered pistol to administer Telazol via a standard Palmer dart. Immobilized bears were transported to a necropsy suite in a nearby building. After blood sampling, bears were euthanised by an intravenous injection of pentobarbital and death by cardiac arrest assessed with a stethoscope. Tissue collection followed immediately with samples frozen in liquid nitrogen and was complete within 15 min.American black bears (31–118 kg) were captured May-July from the field either near McGrath or Anchorage, Alaska. Bears were transferred to Fairbanks, Alaska where they were held individually in a shaded outdoor holding facility. In summer and fall, bears were fed dog chow and had continuous access to water. Bears representing the non-hibernating condition were still feeding and active when they were euthanized and sampled for tissues between late May and late July (n = 6). Food was withdrawn 24 hours before these animals were sacrificed. Bears in the hibernating condition were euthanised for tissue sampling between March 1Animals were anesthetized with carbon dioxide and perfused transcardialy with physiological saline supplemented Heparin and afterwards with 4% paraformaldehyde and 0.1% glutaraldehyde in phosphate buffered saline (PBS). Subsequently the brain was removed, postfixed for 24 h in 4% paraformaldehyde in PBS, and cryoprotected by immersion in 30% sucrose in PBS for 48 h. The brains where cut into 30 µm thick sagittal sections and stored at 4°C in PBS with 0.01% sodium azide added as a preservative. Briefly, free-floating sections were processed for immunoperoxidase labelling: sections were rinsed in PBS, and incubated 30 min in 0.3% hydrogen peroxide in PBS. Sections were rinsed in TBS-T  and incubated for 60 min in 5% normal donkey serum . The primary antibody  was applied overnight. Subsequently, sections were rinsed, incubated 2 h with biotinylated donkey-anti-mouse , rinsed, incubated for 45 min with ExtrAvidin , rinsed, and developed with nickel-enhanced diaminobenzidine. Afterwards the sections were mounted, dehydrated, cleared, and coverslipped.2; 2 mM EDTA; 2 mM EGTA; 1% NP40; 5 mM NaF; 1 mM Na3VO4; 5% glycerol; 1 mM PMSF; 1 µg/ml leupeptin; Complete protease inhibitor cocktail ) using Ultra Turrax. Homogenates were centrifuged , and supernatants were transferred into new tubes.Brain tissue samples were ground in liquid nitrogen, aliquoted and stored at −80°C. Tissue samples for Western blot analysis were homogenised in 9 volumes (w/v) protein extraction buffer B . The immunoreactivity of phosphosite-specific tau antibodies was related to the expression of tau determined using an antibody directed against the non-phosphorylated protein. The uniformity of sample loading was confirmed by immunodetection of beta-actin and Coomassie-stain of the corresponding membrane after stripping.A potential, hibernation-state related alternative splicing of the tau exon 10 was determined in arctic ground squirrels . For this purpose neocortical brain tissue samples (70–100 mg) were homogenised in 1 ml Trizol reagent  and mRNA was isolated according to the instructions of the product supplier. Quality and integrity of isolated mRNA was assessed by electrophoresis. Prior to the expression analysis a RT-PCR using degenerated primer  were separated by SDS-PAGE and transferred to a PVDF membrane (see 2.2.1). The proteins were stained using the Pro-Q Diamond phosphoprotein blot stain kit  according to the instructions of the product supplier.2; 10 mM MnCl2; 80 ng/µl recombinant tau protein; 100 µm ATP [cold]; 50 nCi/µl 32P ATP) and incubated at various temperatures and times. Reactions for the analysis of tau phosphate net turnover were started at 37°C and aliquots were taken successively after incubation at temperatures that were lowered stepwise. To determine the tau phosphate net turnover in an increasing temperature gradient the pre-phosphorylation of tau was performed by 15 min incubation at 30°C and samples were immediately chilled on ice. The temperature was increased stepwise and aliquots were taken successively from the samples after incubation. The record of reaction was started after incubation at 5°C. Reactions were terminated by adding SDS-sample buffer followed by heat denaturation at 95°C for 10 min. Samples were separated by SDS-PAGE; gels were dried and exposed to X-ray film. Optical density was analysed using the 1D module of TotalLab software (Nonlinear Dynamics) based on 16-bit grey-level film scans.To determine the temperature sensitivity of enzyme activity we used cortical brain samples from arctic ground squirrels  that were homogenized with an Ultra Turrax in five volumes of buffer K . Protein concentrations were determined according to Bradford The activity state of the tau kinases glycogen synthase kinase 3 beta (GSK3-beta), cyclin dependent kinase 5 (cdk5), stress-activated protein kinase/Jun-amino-terminal kinase (SAPK/JNK) and mitogen activated protein kinase/extracellular regulated protein kinase (MAPK/ERK), was determined by Western blot as described previously (2.2.1). Immunoreactivities of the phosphosite-specific antibodies were related to the expression of the according proteins determined applying antibodies directed against the non-modified molecule . The conTable S1Spermophilus parryii) and Syrian hamsters (Mesocricetus auratus). The table lists the increase of tau phosphorylation at specific sites in different brain regions during the hibernation cycle . Data are based on Western blot experiments. Increase factors are related to the level of phosphorylation in euthermic animals. One way ANOVA (unblocked) was performed and italic p-values indicate significant alterations (p≤0.05). Newman-Keuls contrasts are listed to indicate the difference in tau phosphorylation level between all analysed groups.Summary of hibernation-dependent tau phosphorylation in arctic ground squirrels ((0.04 MB DOC)Click here for additional data file.


The Hippo/MST1 signaling pathway plays an important role in the regulation of cell proliferation and apoptosis. As a major downstream target of the Hippo/MST1 pathway, YAP2 (Yes-associated protein 2) functions as a transcriptional cofactor that has been implicated in many biological processes, including organ size control and cancer development. MST1/Lats kinase inhibits YAP2's nuclear accumulation and transcriptional activity through inducing the phosphorylation at serine 127 and the sequential association with 14-3-3 proteins. However, the dephosphorylation of YAP2 is not fully appreciated.in vitro and in vivo, and PP1A-mediated dephosphorylation induces the nuclear accumulation and transcriptional activation of YAP2. Inhibition of PP1 by okadiac acid (OA) increases the phosphorylation at serine 127 and cytoplasmic translocation of YAP2 proteins, thereby mitigating its transcription activity. PP1A expression enhances YAP2's pro-survival capability and YAP2 knockdown sensitizes ovarian cancer cells to cisplatin treatment.In the present study, we demonstrate that PP1A  interacts with and dephosphorylates YAP2 Our findings define a novel molecular mechanism that YAP2 is positively regulated by PP1-mediated dephosphorylation in the cell survival. Drosophila and mammals Hippo/MST signaling pathway has been delineated as a tumor suppressor pathway, which has been implicated in the diverse biological processes including cell proliferation, apoptosis, organ size control, and cancer development in both Drosophila and mammals. Yki (Yorkie), the Drosophila orthologue of mammalian YAP2, promotes cell growth through transcriptionally co-activating Sd  YAP2 was initially identified as a Src/Yes kinase associated protein It has long been demonstrated that the post-transcriptional modifications (PTMs) of proteins are important for the regulation of its function, especially for the transcriptional factors and co-factors. The phosphorylation and ubiquitination of YAP2 has been reported The phosphoprotein phosphatase superfamily members include PP1, PP2A (PP2), PP2B (PP3), and PP4–7, among which PP1 is a major eukaryotic Ser/Thr protein phosphatase that involved in a variety of biological processes in vitro and in vivo and that PP1A dephosphorylates YAP2 at serine 127 and dissociates it from 14-3-3 binding, thus leading to its nuclear retention and transcriptional activation. We also observed that YAP2 expression inhibits ovarian cancer cell death induced by cisplatin treatment and the inhibition of PP1A with okadiac acid increases the phosphorylation and cytoplasmic translocation as well as the drug resistance capability of YAP2. Our findings demonstrate that YAP2 is functionally regulated by PP1A-mediated dephosphorylation.In this study, we identify PP1A, as a component of YAP2 protein complex, regulates YAP2 function in the DNA damage-induced cell death. We found that PP1A and YAP2 interact with each other To gain the further understanding of molecular mechanism underlying YAP2 regulation, we undertook the biochemical purification of YAP2. We generated stable HeLa cells expressing human YAP2 tagged with both the FLAG and haemagglutinin (HA) epitopes at its amino terminus. Cytoplasmic and nuclear extracts from these cells were subjected to sequential purification with anti-FLAG and anti-HA antibody columns. As shown in To further define PP1A as a YAP2 complex protein, we performed glycerol-gradient sedimentation and gel-filtration experiments. PP1A sedimented together with YAP2 . Upon exin vitro kinase assay by incubating the recombinant YAP2 proteins with the immunoprecipitated Lats2 kinase, followed by in vitro dephosphorylation assay using recombinant GST fusion protein encoding PP1A. We found that Lats2 phosphorylated YAP2 at serine 127 and PP1A dephosphorylated it  is one of the putative YAP2-TEAD target genes CTGF expression in quantative-RT-PCR assays  supplemented with 10% fetal bovine serum (Invitrogen), 100 U/ml penicillin and streptomycin (Gibco). Antibodies of FLAG (Sigma), Myc (Santa Cruz), PP1A (Epitomics), GST (Santa Cruz), YAP2 (Santa Cruz), Phospho-Ser127-YAP2 , Erk1/2 , Actin (SIGMA), GAPDH (SIGMA) were purchased. Cisplatin, okadaic acid and CHX (Cyclohexamine) were purchased from Sigma.HeLa cells and Ovarian cancer cells (ATCC) were cultured at 37°C and 5% CO3xFLAG- tagged MST1 construct was subcloned in the pCMV10 expression vector. Myc-tagged YAP2 and Myc-tagged Lats2 constructs were subcloned into the pCMV-Myc expression vector. GFP-YAP2 construct is in the GFP-C2 expression vector and GST-YAP2 and GST-PP1A construct is in the pGEX expression vector. Ad-YAP2, Ad-GFP, Ad-YAP2 shRNA or Ad-U6 adenovirus was generated to infect the cells. YAP2 shRNA targeting sequence: GACAUCUUCUGGUCAGAGA 2) containing protease and phosphatase inhibitors. Cells were homogenized and the nuclear or cytoplasmic fractions were collected respectively. YAP2 was immunoprecipitated from the nuclear or cytoplasmic extracts by using the anti- FLAG and HA beads sequentially and the final eluate was digested with trypsin and analyzed by mass spectrometry.TAP assay has been previously described The precipitated proteins from TAP were centrifuged in 10%–40% gels as described Immunoprecipitation and immunoblotting analysis was carried out as described previously. Briefly, cell lysates were incubated with the indicated antibodies in the presence of 15 µL of protein A-protein G (2∶1)-agarose beads for 2 hours at 4°C. After washed four times, the immunoprecipitates were subjected to electrophoresis. Protein expression was examined by probing Western blots of total cell lysates or immunoprecipitates with the appropriate antibodies as noted in the figure legends. Detection of band intensity was carried out with the ECL Western blotting Analysis System.In vitro kinase reactions were carried out by incubating immunprecipitated Lats and the recombinant GST-YAP2 for 30 min at 30°C in the presence of 3 µM cold ATP in 30 µl of buffer containing 20 mM HEPES (pH 7.4), 10 mM MgCl2, 10 mM MnCl2, 1 mM DTT In vitro dephosphorylation assay was performed by incubating the kinase reaction mixture with the recombinant GST-PP1 or GST only protein for 1 hour at 37°C in buffer containing 50 mM HEPES, 100 mM NaCl, 1 mM MnCl2, 2 mM DTT, 0.1 mM EGTA and 0.025% Tween 20 followed by the SDS-PAGE gel and immunoblotting with the indicated antibodies.Luciferase activity was measured as described Total RNA was isolated from cultured cells using Trizol reagent (Invitrogen). cDNA was synthesized by reverse transcription using oligo dT as the primer and proceeded to real-time PCR with gene-specific primers in the presence of SYBR Premix Ex Taq . The relative abundance of mRNA was calculated by normalization to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. Primers sequence used for CTGF is 5′-gcttaccgactggaagacacg and 3′- cggatgcactttttgccctt.Cell death assays were performed according to the manufacturer's guidelines (BD Pharmingen). Briefly, cells were cultured in 6-well plate and grown in DMEM medium supplemented with 10% fetal bovine serum for 24 hours, then treated with cisplatin (20 µM) for 24 hours, both floated and attached cells were collected. Cells were washed with PBS, and resuspended in binding buffer containing 5 µL Annexin V followed by flow cytometry (Beckman Coulter). All the experiments were performed three times.t-test. Data are presented as the mean ± SEM except for analyses of luciferase assays where mean ± SD is shown. *P <0.05 or **P<0.01 denotes statistical significance.Statistical analysis of the data was performed with a two-tailed Student's


Here we demonstrate that resveratrol acutely reduces amiloride-sensitive current in mpkCCDc14 cells. The time course and dose dependency of this inhibition paralleled depletion of the PIP3 reporter (AKT-PH) in live-cell microscopy, indicating the early inhibition is likely mediated by resveratrol's known effects on PI3K activity. Additionally, resveratrol induces a late inhibitory effect (4–24 hours) that appears to be mediated via AMPK activation. Resveratrol treatment induces significant AMPK activation compared with vehicle controls after 4 h, which persists through 16 h. Knockdown of AMPK or treatment with the AMPK inhibitor Compound C reduced the late phase of current reduction but had no effect on the early inhibitory activity of resveratrol. Collectively, these data demonstrate that resveratrol inhibits ENaC activity by a dual effect: an early reduction in activity seen within 5 minutes related to depletion of membrane PIP3, and a sustained late (4–24 h) effect secondary to activation of AMPK.Resveratrol, a naturally occurring phytoalexin, has reported cardioprotective, anti-inflammatory, chemopreventative and antidiabetic properties. Several studies indicate the multiple effects of resveratrol on cellular function are due to its inhibition of class 1A phosphoinositide 3-kinase (PI3K) mediated signaling pathways, but it also activates AMP-activated protein kinase (AMPK). As sodium transport in the kidney via the Epithelial Sodium Channel (ENaC) is highly sensitive to changes in phosphoinositide signaling in the membrane and AMPK, we employed resveratrol to probe the relative effects of phosphatidylinositol species in the plasma membrane and AMPK activity and their impact on ENaC activity in mouse cortical collecting duct (mpkCCD Several mechanisms of hormone-induced sodium transport involve phosphoinositide (PI) synthesis and metabolism and thus intersect with the trafficking pathways that alter surface density of ENaC. Indeed, studies from our laboratory and others support the hypothesis that modulation of the kinases that alter the compartmentalization of PIs may stimulate channel insertion and regulate channel endocytosis.The epithelial Na22 and PIP3. Phosphatidylinositide 3-OH kinase (PI3-K) is a critical component of many signaling pathways regulating ENaC activity. Aldosterone and insulin have both been shown to increase the activity of PI3-K, resulting in activation of signaling pathways that lead to an increase in ENaC expression at the apical membrane 2 and PIP3 can directly bind to ENaC and influence channel open probability, Po, another mechanism of altering overall ENaC function We have demonstrated that ENaC is retrieved into the cell as a ubiquitinated protein via clathrin-mediated endocytosis with epsin as the adaptor protein 2, PIP3 and/or AMPK levels. We have used the agent to probe the intersection of these pathways in mpkCCDc14 cells, a native ENaC culture model. We describe a complex but profound response, mediated initially through effects on PI3-K and a sustained later effect mediated in part by activation of AMPK. These effects may contribute to the antihypertensive effects of resveratrol recently described in animal models Interestingly, resveratrol is also thought to have cytoprotective and anti-aging effects that are mediated via activation of the metabolic sensor, AMP-activated protein kinase (AMPK) 2 reporter, the PH domain from PLCδ1 cloned into pEGFP-N1, was the generous gift of Dr. T Balla . The reporter Akt-PH-11 was kindly provided by Dr. J. Stockand .All reagents and chemicals used were purchased from Sigma unless otherwise noted. Phospho-Thr172-AMPKα antibody was purchased from Cell Signaling Technology , AMPKα1 antibody was obtained from Upstate Millipore. Compound C (6-[4-[2-(1-Piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolopyrimidine) was obtained from Enzo Life Sciences . HRP-conjugated anti-mouse and anti-rabbit antibodies were purchased from GE Healthcare . The PIPc14) cells obtained from Alain Vandewalle (INSERM)Mouse cortical collecting duct  were measured with an automatic voltage clamp. Cells were recorded approximately 30 min prior to addition of resveratrol or vehicle control to apical and basolateral cell surfaces. The scI tracing was recorded for an additional 45 minutes. Amiloride (10 µM) was added at the end of each experiment to derive the amiloride-sensitive component of the short circuit current. Typically, ≥90% of the total Isc was inhibited after amiloride addition. The bathing Ringer's solution was composed of 120 mM NaCl, 25 mM NaHCO3, 3.3 mM KH2PO4, 0.8 mM K2HPO4, 1.2 mM MgCl2, 1.2 mM CaCl2, and 10 mM glucose. The chambers were constantly gassed with a mixture of 95% O2, 5% CO2 at 37°C, which maintained the pH at 7.4. To determine the effect of resveratrol treatment for up to 24 hours, transepithelial Na+ current across mpkCCDc14 cell monolayers cultured on 6.5-mm permeable supports  and kept in culture media was calculated using Ohm's law as the quotient of transepithelial potential difference to transepithelial resistance under open circuit conditions using an EVOM volt-ohm meter with dual Ag/AgCl pellet electrodes  to measure voltage and resistance.mpkCCDGCTGTGGCTCACCCAATTATTCAAGAGATAATTGGGTGAGCCACAGCTTTTTCGATCCCC) at nucleotide 517 , along with a control hairpin that does not target any known mammalian gene (GCGCGCTTTGTAGGATTCGTTCAAGAGACGAATCCTACAAAGCGCGCTTTTTCGATCCCC) (MamX). The oligonucleotides were annealed and first ligated into the pEN vector  and then transferred to the shRNA-expressing lentivirus vector pDSL_hpUGIP (ATCC) by LR-clonase (Invitrogen). Recombinant lentiviruses were generated by co-transfection of these individual plasmids with a mixture of packaging vectors  into HEK 293T packaging cells using Lipofectamine 2000. Viral supernatants were harvested 72 h after transfection. Viral particles (∼1×107/prep were concentrated using an Amicon Ultra 50 filter unit (Millipore #UFC905024). mpkCCDc14 cells grown in a 6-well plate were transduced with lentiviral particles at a multiplicity of infection of ∼1. After 2 weeks of selection in puromycin (2 µg/ml), cells were expanded and plated on 0.33-cm2 Costar Transwell filters (#3470) and allowed to grow to high resistance before use in experiments 5–9 d later.Two complementary primers were used to generate a hairpin targeting mouse AMPK-α1 , or β-actin (1∶5000) primary antibodies. Secondary donkey anti-rabbit or sheep anti-mouse antibodies were used at 1∶5000. Intensities of all relevant bands corrected for local background intensity on the blot were quantitated using a VersaDoc Imager with Quantity One software (Bio-Rad). To measure AMPK activation in the cell lysates, the pThr172-AMPKα band signal in each lane was normalized to the intensity of β-actin as a loading control.mpkCCDc14 cells were seeded at 30% confluence onto 0.17-mm Delta T glass-bottomed dishes  and cultured for 16–24 h to allow cells to adhere. The cells were then transferred to unsupplemented Opti-MEM media (Invitrogen) and immediately transfected with 1.5 ug DNA and Lipofectamine 2000™ reagent (Invitrogen) according to the manufacturer's instructions. After 5-h incubation at 37°C, the transfection solution was replaced with DMEM/F12 medium and cells were returned to incubation. Cells were imaged 24–26 h post-transfection. Transfected cells were imaged in Opti-MEM media supplemented with 5% FBS  and 40 mM HEPES and maintained at 35°C during imaging. Epiflourescent time-lapse images were taken on an Olympus IX81 microscope  equipped with a RETIGA EXI (QImaging) and XENON lamp (Olympus) with appropriate filter combinations for GFP. Time-lapse images were taken with a PlanApo 60× oil immersion objective (1.4 NA) and Metamorph imaging software. Images were taken at 5-min intervals for 30–90 min. Resveratrol was added directly to the imaging media to a final concentration of 100–200 µM, with maximal effect at 200 µM. Images were exported in a tag-information-file format (TIFF), and contrast was adjusted in Adobe® Photoshop® . Image analysis was performed using ImageJ as described For transfection, mpkCCDt tests, as described in each figure legend. In all cases, p values <0.05 were considered significant.Statistical analyses were performed using StatView . Analysis of variance was used to compare data obtained from different batches of cell monolayers for short circuit current experiments. For other biochemical experiments, statistics were performed using Student's c14 cells mounted in a modified Ussing chambers following treatment with increasing concentrations of resveratrol or vehicle in + transport via ENaC. Resveratrol inhibited Na+ current via ENaC in a dose-dependent manner, with an IC50 does of 107 µM,  causes a rapid redistribution of PI3 away from the plasma membrane, as compared with control vehicle-treated cells (lower panels). Vehicle treatment was used to evaluate decreases in fluorescence emissions over time due to photobleaching. Furthermore, the observed change in mean pixel intensity of GFP-AKT-PH at the plasma membrane parallels the acute change in short circuit current .Resveratrol has been demonstrated to inhibit PI3-K by competing with ATP for the catalytic site  current Figure 2c14 cells were fed with media containing resveratrol or vehicle control (ethanol) for 16 h. At various time intervals cell extracts were examined by immunoblotting for phosphorylated AMPKα (p-AMPKα) to measure activation of AMPK in the presence or absence of resveratrol. Resveratrol treatment induced a significant activation of AMPK compared with vehicle controls beginning at 4 h post-treatment and continued for at least 16 h.  for 30 min to allow for AMPK inhibition before resveratrol treatment (c14 cells but did not affect the early (0–6 h) inhibitory effect of resveratrol. Current values for control cells (no resveratrol) with or without Compound C were not statistically different. These results demonstrate that acute inhibition of AMPK abrogates the late inhibitory effect of resveratrol on ENaC current.To investigate whether the late inhibitory effect of resveratrol is due to AMPK activity, mpkCCDreatment Figure 6c14 cells by RNA interference and examined the effects of resveratrol. Knockdown of AMPKα1 was confirmed by immunoblotting for AMPKα1 expression  but did not affect the early (0–4 h) inhibitory effect of resveratrol,  at the apical membrane, and open probability (oP) by a variety of hormonal and cellular regulatory pathways (reviewed in oP via a direct interaction with the channel. Inhibition of PI3-K activity in ENaC-expressing cells has been shown to decrease channel activity in parallel with decreases in ENaC expression at the plasma membrane N at the apical membrane by regulation of the ubiquitin ligase Nedd4-2 and stimulating retrieval of the channel from the membrane c14 cells in a rapid and sustained manner. The early effects appear to be mediated by PI3-K inhibition while the sustained effects require the activation of AMPK.The epithelial sodium channel, ENaC, is tightly regulated in both number (c14 cells resulted in a rapid and dose-dependent decline in Na+ current over a period of minutes ( and + current was reduced by greater than 50% within 5-10 minutes of resveratrol addition and remained suppressed for a period of hours as long as resveratrol remained present ( and 3 levels observed via live-cell microscopy (3 levels and Na+ current following addition of resveratrol is consistent with the inhibition of PI3-K activity described in other tissues 3 on maintaining ENaC activity under basal culture conditions in CCD cells. The studies documented here are consistent with this being a plasma membrane pool of PI 3, but cannot rule out the potential for changes in sub-membrane pools of PI 3 participating in this phenomenon. The rapidity of the decline in PI 3 levels further suggests that it is maintained at the membrane by a dynamic balance between kinase and phosphatase activity, as previously suggested Addition of resveratrol to mpkCCDtes  and 2. Na+ c14 cells. Given our model that resveratrol inhibits ENaC in the early phase via a mechanism different from AMPK, we would anticipate the inhibitor of AMPK, compound C, would not interfere with the early inhibition. Indeed, as seen in andThe ENaC inhibition seen with resveratrol is both rapid in onset and sustained for prolonged periods. To examine the sustained inhibition of ENaC further, we looked for effects of resveratrol on AMPK activity, a known long-term regulator of ENaC activity + handling mediated through inhibition of ENaC by both PI3-K and AMPK pathways. Clearly, other pathways also must be involved, since cardioprotective effects and vascular effects of resveratrol have been described in animal models where hypertension is not affected by this agent We propose that the renal and cardioprotective effects of resveratrol described in hypertensive animals may be, to some degree, related to altered Na