These observations were confirmed by anti-acetyl-lysine immunoblotting. In summary, cardiac lysine acetylation may play a role in cardiac substrate selection, bioenergetic performance, and maintenance of redox balance. New sites suggest a host of potential mechanisms by which excitation-contraction coupling may also be modulated.In the heart, lysine acetylation has been implicated in processes ranging from transcriptional control of pathological remodeling, to cardioprotection arising from caloric restriction. Given the emerging importance of this post-translational modification, we used a proteomic approach to investigate the broader role of lysine acetylation in the heart using a guinea pig model. Briefly, hearts were fractionated into myofilament-, mitochondrial- and cytosol-enriched fractions prior to proteolysis and affinity-enrichment of acetylated peptides. LC-MS/MS analysis identified 1075 acetylated peptides, harboring 994 acetylation sites that map to 240 proteins with a global protein false discovery rate <0.8%. Mitochondrial targets account for 59% of identified proteins and 64% of sites. The majority of the acetyl-proteins are enzymes involved in fatty acid metabolism, oxidative phosphorylation or the TCA cycle. Within the cytosolic fraction, the enzymes of glycolysis, fatty acid synthesis and lipid binding are prominent. Nuclear targets included histones and the transcriptional regulators E1A(p300) and CREB binding protein. Comparison of our dataset with three previous global acetylomic studies uniquely revealed 53 lysine-acetylated proteins. Specifically, newly-identified acetyl-proteins include Ca Recently, the advent of new proteomic tools has permitted global scale assessments of lysine acetyl-proteomes Acetylation of lysine residues on histones was first recognized as a post-translational modification nearly 50 years ago In cardiac biology, histone acetylation is a mediator of the transcriptional programs that underlie cardiomyocyte proliferation ad libitum. This study conforms to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health  and was approved by the Johns Hopkins Animal Care and Use Committee.300 g male Hartley guinea pigs were obtained from Hill Top and housed in an animal facility at The Johns Hopkins University where they had access to a standard chow diet and drinking water 3 in a petri dish containing 5 mL of isolation buffer. The mince for each heart was rinsed twice with isolation buffer and homogenized in isolation buffer supplemented with lysine deacetylase inhibitors, Trichostatin A (1 µM), sirtinol (85 µM), nicotinamide (5 mM), and splitomycin (170 µM), using a chilled Potter-Elvehjem homogenizer . Homogenates were centrifuged at 700×g for 10 min. The supernatant was set aside and the pellet was resuspended in isolation buffer to extract trapped mitochondria before a second round of centrifugation. The resulting pellet, containing the bulk of the myofilaments, nuclei, and residual mitochondria, was snap-frozen in a dry ice/ethanol bath and frozen immediately at −20°C. The supernatants were combined and centrifuged at 8,000×g for 20 min. The pellet containing crude mitochondria and associated plasma membranes and sarcoplasmic reticulum was snap frozen as above. The supernatant was centrifuged at 100,000×g for 1 hr. The pellet and the supernatant (cytosol) were likewise snap frozen and stored at −20°C until further use.Three guinea pig hearts were isolated and perfused with ice-cold isolation buffer before being minced into pieces about 2–3 mmet al. ave) in a SW41-Ti rotor for 18 hours at 4°C to resolve the mitochondrial respiratory complexes. Samples were recovered by puncturing the bottom of the polyallomer centrifuge tube with a 23-gauge needle and collecting 1.5 mL aliquots, drop-wise.Mitochondrial complex I and complex V were enriched from mitochondria by sucrose density gradient as described by Foster 2 gas over the samples for 1–2 minutes. The precipitated proteins were re-dissolved in 10 mM HEPES, 6 M urea, 2 M thiourea, pH 8.0. After 4-fold dilution in deionized water such that the final concentrations of urea and thiourea were 1.5 M and 0.5 M respectively 2HPO4. Immunoprecipitation was carried out as essentially described by Choudhary and colleagues °C on a rotation wheel. The immunoprecipitates were washed 4 times with the immunoprecipitation buffer followed by two washes with distilled water. Residual water was removed and acetylated peptides bound to antibodies were eluted in 0.1% formic acid.Twelve samples, representing the 4 subcellular components from each heart, were delipidated and precipitated and concentrated by methanol/water/chloroform precipitation as described Wessel and Flugge 6, while the ion trap MS2 scans were performed at a target value of 10,000 ions. Maximum injection times were both set to 100 ms. The ion selection threshold was set to 2,000 counts and an isolation width of 1.9 daltons was used to perform CID fragmentation with a normalized collision energy of 35%. Ambient polysiloxane produced a background peak at 371.101230 m/z, which was used as an internal calibrant for each survey scan.Peptides were injected onto a 2 cm trap column at 5 µL/minute for 6 minutes before being eluted onto a 75 µm×15 cm in house packed column  using a nanoAquity nanoLC system (Waters) operating at 300 nL/min. Each sample was run on a 90-minute gradient with double sawtooth cleanup gradients between each run. The peptides were eluted and ionized into an Orbitrap Velos mass spectrometer (Thermo Fisher) at 2.0 kV using a data-dependent “Top 20” method operating in FT-IT parallel acquisition mode. The survey full-scan MS (m/z from 350–1700) was performed at a resolution 60,000 with a target of 1×10Peak list files (.RAW) were searched against a guinea pig database of predicted proteins , using Mascot Version: 2.2.0 (Matrix Science). Spectra were searched with a mass tolerance of 15 ppm in MS mode and 0.8 Da in MS/MS mode. Trypsin was specified as the enzyme and 4 missed cleavage sites were allowed. Cysteine carbamidomethylation was searched as a fixed modification, whereas N-pyroglutamine, oxidized methionine and acetylation of lysine was searched as variable modification. All searches were conducted with the reversed-database search mode engaged. Mascot output files (.dat) were imported into Scaffold 3Q+ (v. 3.1.2), where spectra were also searched against the same guinea pig database, using identical search parameters, with X!Tandem et al. Peptide identification probability was assessed by PeptideProphet http://blast.ncbi.nlm.nih.gov/Blast.cgi).To facilitate comparisons with other datasets, the locations of acetylation sites within the guinea putative pig primary sequences were mapped to the corresponding position within human homologs, using the Basic Local Alignment Sequence Tool, blastp  at 150 V for 35 mins (MES buffer) or 50 mins (MOPS buffer). Proteins were transferred to nitrocellulose with the i-Blot system (Invitrogen) using a 9-minute transfer. Blots were blocked with 5% (w/v) bovine serum albumin (BSA)  in Tris-buffered saline containing 0.1% (v/v) Tween-20 for a minimum of 1 hour. Primary anti-acetylated lysine antibodies , were incubated in Tris-buffered saline  supplemented with 0.1% (v/v) Tween-20 (TBS-T) containing 1% (w/v) BSA overnight at 4°C. Control blots were conducted by preincubating acetyl-lysine antibodies with acetylated BSA  2+) Structural models were generated using the web-based modeling suite, Swiss-Model  of identified acetyl-proteins harbored more than one acetylation site. In nd entry), 5 of the 25 acetylation sites account for 62% of the site-counting spectra. The relative intra-protein frequency of site detection among these multi-acetylated proteins is a cryptic metric. It may provide a crude first approximation of relative site occupancy. However, intrinsic peptide properties, including ionizability, fragmentation susceptibility, and affinity for the acetyl-lysine antibody, are potential confounding factors. Limitations notwithstanding, the site identification frequency on a heavily targeted protein may help prioritize the design of mutants and is, therefore, provided in We also note that among the most heavily acetylated proteins (e.g. top 10), the distribution of assigned MS/MS spectra is often heavily weighted toward a much smaller subset of sites. For example, though myosin heavy chain acetylation was detected on 49 sites, 10 sites account for 72% of the site-counting spectra for that protein. Similarly, for Trifunctional Protein, alpha subunit  and Complex V  displayed immunoreactivity toward both monoclonal and polyclonal anti acetyl-lysine antibodies on multiple subunits. The immunoreactivity of myofilaments, complex I and complex V were all diminished by performing the blots in the presence of competing acetylated BSA (>2% w/v), which confirmed the specificity of the antibodies for acetylated lysine residues.2+ fluxes, i.e., the relative amount of Ca2+ entering and leaving the cell on each heartbeat versus that released and recycled through intracellular Ca2+ stores is close to that of larger animals and humans 2+ handling properties that make it difficult to extrapolate findings to the human disease process. Other large animals, such as the rabbit, dog or cat could also provide relevant information; however, at much greater expense. Another major advantage is that the basic properties of guinea pig cardiac physiology have been extensively characterized previously, and the aortic banding model of hypertrophy and failure has also been validated in prior studies, including changes occurring in ion channels and Ca2+ handling that are similar to findings in human failing hearts. An additional advantage is that the computational models of the guinea pig cardiomyocytes we have developed are the most comprehensive of any species available, and the only ones that incorporate energetics and ROS metabolism For this proteomic study, we chose the guinea pig model since aspects of its cardiac physiology more closely approximate humans, than do rat or mouse hearts. Specifically, unlike other small animal models such as the rat, mouse, or hamster, the guinea pig has an action potential profile that displays a long plateau and a complement of ion channels and exchangers that is very similar to that of humans. In addition, the waveform of the guinea pig electrocardiogram is similar to humans At the outset of the project, predicted or confirmed guinea pig protein sequences were scarce in prominent protein databases . However, proteomic analysis was possible using the predicted protein sequences obtained from the whole guinea pig genome. The Ensembl CavPor3.59 database contains 19,744 predicted protein sequences, enough to minimize the likelihood of failing to identify key proteins. To our knowledge this is one of few large-scale proteomic studies conducted in the guinea pig model system The present work revealed the diverse nature of acetylation targets in the guinea pig heart and identified a large number of novel targets for which functional assessment is warranted in the future. GO analysis revealed that acetylated proteins from mitochondrial ontologies including lipid metabolism, redox balance and ATP synthesis were more prominent than expected by chance . AnotherRecent work on lysine acetylation has shown that the enzymes of metabolism are prime targets from prokaryotes 46 and K101. However, given that our gel-free strategy identified at total of 163 sites on multiple subunits of each of the respiratory complexes ; MDH2; mCK(K344); mouse sequence numbering), were corroborated in the guinea pig dataset.We detected many previously reported acetyl-protein targets in the fatty acid oxidation pathway, TCA cycle and electron transport chain in our survey. In general, we significantly extended the number of sites modified/protein as well as the number of subunits modified per multiprotein complex. For example, for the electron transport chain, we found 29 sites on 14 subunits of complex I and 64 sites on 8 subunits of the ATP synthase. In other cases, like the A subunit of succinate dehydrogenase, acetylation at Kunit cf. . Acetyla2.−) dismutase 1 (SOD1) and 2 (SOD2), and the thiol protective antioxidant enzymes, thioredoxin and the peroxiredoxins. Deacetylation of mitochondrial MnSOD (SOD2), mediated by SIRT3 2.− -scavenging activity and decreases overall ROS generation in response to caloric restriction 68 and K122) We note that enzymes of antioxidant defense and redox regulation are affected by acetylation. This group includes the ROS scavenger enzymes, superoxide  harnesses the mitochondrial proton-motive force to maintain matrix NADPH levels. Other major sources of NADPH include isocitrate dehydrogenase 2 (IDH2) and malic enzyme (ME). Both NNT and IDH2 are among the top 40 most heavily acetylated proteins , and haret al. Nuclear acetyl-proteins were found in our myofilament-rich fraction. Among them was p300, the transcriptional co-activator with histone acetyltransferase activity. Using our global approach, we confirmed that all 9 of the autoacetylation sites, within its flexible activation loop, that were previously identified in an intensive study of bacterially overexpressed p300 2+ release from sarcoplasmic reticulum (SR) stores, is acetylated at K1087 (human K1141), in its cytoplasmic foot structure, specifically within a domain designated SPRY-2. The precise role of the SPRY-2 domain in RyR function is unclear, though it has been postulated that it may participate in both inter- and intra-molecular protein interactions The most unique aspect of the cardiac lysine acetylome is the observed modification of proteins involved in excitation-contraction coupling. Among them, the cardiac ryanodine receptor, RyR2, responsible for Ca2+ from the cytoplasm, back into the SR, is performed by the sarcoplasmic/endoplasmic reticulum Ca2+/ATPase, of which the SERCA2a isoform is found in the heart. Here we show that guinea pig SERCA2 is acetylated at 3 sites, all of which reside in the cytoplasmic nucleotide-binding domain (N-domain). Preliminary structural modeling of the guinea pig sequence and sites using the rabbit structure  442, F487 and K515) 464, K510, K533) lie on the protein surface. However, both K510 and K533 do lie on the opposite end of the same beta sheet as critical residue K515, which begs the question of whether the structural consequences of acetylation at these surface sites might be transduced inward and thereby perturb the adenine-binding site.Sequestration of Ca413, lies within the so-called hypertrophic cardiomyopathy (HCM) loop that forms part of myosin’s actin-binding interface. Indeed, several lysines acetylated in our study are found in key functional domains. For instance, K383 lies near the distal end of myosin where the head interacts with actin, and its mutation has been associated with HCM (K383N) 246, an HCM locus (K246Q) th beta strand of the central beta sheet known as the transducer domain, which couples movements between the actin- and ATP-binding domain sites 246 may recapitulate the myosin and cardiac dysfunction conferred by the K246Q mutation. Other notable acetylation sites include K434 adjacent to HCM mutation site, M435, in the long helix at the tip of the myosin head, and K744 adjacent to E743 in the converter domain, which serves to amplify small conformational changes in the myosin head into large swings in the myosin lever arm that accompany myosin ATPase activity and drive muscle contraction. Intriguingly, whether lysine acetylation adjacent to HCM mutations might contribute to phenotypic variability observed among HCM patients has not been investigated.Our studies show sarcomeric acetylation at the level of both the thick and thin filaments. Thick filament myosin is heavily acetylated at up to 49 sites . Just ovin vitro motility assays, and levels of myosin acetylation increased when cardiac hypertrophy was induced by thoracic aortic constriction in mice. In vitro, myosin is acetylated by PCAF at K549 and K633, though only K633 was identified from cardiac myocytes cultured in the presence of deacetylase inhibitors and acetyl-CoA. Neither site was among the many identified in our study, though we would not dispute the previous acetylation site assignments, as there are substantial differences in the methodology employed. Rather, our data, taken together with prior studies of myosin acetylation, simply point to the need for rigorous site-by-site determination of in vivo acetylation stoichiometry.The effects of lysine acetylation on myosin have been investigated recently 46 and K165. The primary function of the myosin light chains (MYL2 and MYL3) is to bind and provide stability to the myosin lever arm. In mice, K46 is conserved among the atrial, ventricular, skeletal and smooth muscle isoforms, whereas K165 is found only in the striated muscle isoforms. K46 lies between D45 and D48, both of which are key Ca2+/Mg2+-coordinating residues of the EF hand domain in MYL2 48 by mutation to alanine is sufficient to ablate Ca2+-binding 46 within the metal-binding loop, appreciable levels of acetylation have the potential to alter myofilament kinetics and cardiac function.We also show that acetylation sites are found in the in the N- and C-terminal regions of the myosin regulatory light chain (RLC), MYL2, at K2+-dependent tropomyosin movement over actin 36, is a residue whose mutation causes dilated cardiomyopathy (K36Q) 193, is adjacent to R192, whose mutation confers restrictive cardiomyopathy  2+-sensitivity and cooperativity of Ca2+ activation. Ultimately, the extent of these effects would depend on the acetylation stoichiometry.The thin filaments consist of filamentous actin, lined with the regulatory proteins, tropomyosin, and the three proteins that comprise the troponin complex, Troponins C, I and T. In this study, each of these thin filament proteins is acetylated, and two of them, cardiac troponin I (cTnI) and tropomyosin, harbor 8 and 7 sites, respectively. In cTnI, 3 sites lie within the N-terminal half of the molecule in regions that bind Troponin T as well as the C-terminal lobe of TnC. 3 more sites, are found within the C-terminal mobile domain, near the so-called “second-actin binding site” and in the C-terminus region responsible for proper CaHere, we present the first glimpse of the guinea pig cardiac lysine acetylome. The dataset may not necessarily be comprehensive, as it is subject to any biases that may arise from the specificity of the acetyl-lysine antibody chosen for affinity-peptide enrichment, though this strategy was recently used to identify as many as 15,474 sites from 16 rat tissues Finally, from the proteomic data we have extracted working hypotheses by which lysine acetylation may affect the function of several novel targets and have an impact on excitation-contraction coupling. Developing these hypotheses further, however, will require detailed assessments of acetylation site stoichiometry. Moreover, if acetylation of these targets does regulate their function, over what time scale does it occur? Indeed, the dynamics of acetylation in the heart have not yet been fully addressed, nor has the extent of crosstalk with other lysine modifications, including ubiquitination, sumoylation and methylation among others. There have been reports of lysine acetylation interfering with serine phosphorylation, particularly where the acetylated lysine is immediately N-terminally adjacent to a serine (K(Ac)S) Table S1PTM Count & Site Probability, Spectrum Report, Peptide Report. Sheet 1 shows the sites of acetylation, the statistical probability that the acetyl group can be assigned to a specific lysine residue (in lower case k), and the the number of times that a site was identified in each of 24 LC-MS/MS runs. Sheet 2 provides spectrum data obtained for all acetylated peptides. Sheet 3 includes the data for all peptides, including unacetylated peptides that also eluted from the immunocapture resin. Sheet 4 shows the acetylated peptides in which the site of acetylation (k) lies N-terminally adjacent to serine.(XLSX)Click here for additional data file.Table S2Acetyl-proteins & Sites. Sheet 1: Additional annotation is provided for et alet alet al(XLSX)Click here for additional data file.Table S3BINGO Analysis. Sheet 1: Gene set enrichment of ontologies related to biological processes. Sheet 2: Gene set enrichment of ontologies related to molecular function. Sheet 3: Gene set enrichment of ontologies related to cellular components. These Tables include only terms deemed statistically significant (p<0.01) after Benjamini-Hochberg correction for multiple-hypothesis testing.(XLSX)Click here for additional data file.Table S4Intraprotein Site Identification Frequency. For each acetylation site identified, the number of spectra implicating that site is expressed as a percentage of the total number of spectra attributed to all acetylation sites within a given protein. This provides an indication of the relative frequency with which each site on a protein was identified. Despite confounding factors discussed in the text, it may provide a tool to help prioritize the construction of site-mutants for proteins with many acetylation sites.(XLSX)Click here for additional data file.


Severe burn injury results in the loss of intestinal barrier function, however, the underlying mechanism remains unclear. Myosin light chain (MLC) phosphorylation mediated by MLC kinase (MLCK) is critical to the pathophysiological regulation of intestinal barrier function. We hypothesized that the MLCK-dependent MLC phosphorylation mediates the regulation of intestinal barrier function following burn injury, and that MLCK inhibition attenuates the burn-induced intestinal barrier disfunction.In vivo intestinal permeability to fluorescein isothiocyanate (FITC)-dextran was measured. Intestinal mucosa injury was assessed histologically. Tight junction proteins ZO-1, occludin and claudin-1 was analyzed by immunofluorescent assay. Expression of MLCK and phosphorylated MLC in ileal mucosa was assessed by Western blot. Intestinal permeability was increased significantly after burn injury, which was accompanied by mucosa injury, tight junction protein alterations, and increase of both MLCK and MLC phosphorylation. Treatment with ML-9 attenuated the burn-caused increase of intestinal permeability, mucosa injury, tight junction protein alterations, and decreased MLC phosphorylation, but not MLCK expression.Male balb/c mice were assigned randomly to either sham burn (control) or 30% total body surface area (TBSA) full thickness burn without or with intraperitoneal injection of ML-9 (2 mg/kg), an MLCK inhibitor. The MLCK-dependent MLC phosphorylation mediates intestinal epithelial barrier dysfunction after severe burn injury. It is suggested that MLCK-dependent MLC phosphorylation may be a critical target for the therapeutic treatment of intestinal epithelial barrier disruption after severe burn injury. It is well known that the intestinal epithelial mucosa plays a pivotal role in the host's protection against luminal pathogens and antigenic molecules, providing a barrier function to protect against the invasion of intraluminal microorganisms and endotoxin through the intestinal wall into the blood or lymph. However, it is also well documented that the intestinal epithelial barrier function is often disrupted in many surgical diseases, including trauma, shock, burn injury, and the other surgically critical illness, resulting in the increased intestinal permeability and subsequent translocation of bacteria or/and endotoxin from gastrointestinal tract in vitro and in vivo.An intact intestinal barrier is maintained through tight junctions, which are the key elements of the paracellular space, and intercellular junctions, which assist to seal the paracellular space between adjacent intestinal epithelial cells To date, the mechanisms contributing to intestinal barrier breakdown after severe burn injury have not yet been elucidated. However, a recent study has shown that pentoxifylline, a nonspecific phosphodiesterase inhibitor known to increase intracellular levels of cyclic AMP, is able to limit burn-induced intestinal barrier breakdown and intestinal inflammation, and to attenuate MLCK activation The animal studies were approved by the Animal Care and Use Committee of the Third Military Medical University, and all the protocols were approved by the Ethics Committee of Southwest Hospital, Third Military Medical University, Chongqing, China.Healthy adult male balb/c mice weighing 20–25 g, supplied by the Animal Center, Third Military Medical University, were used in this study. The animals were housed in wire-bottomed, wire-lid cages, allowed access to chow and water ad libitum, and acclimatized for 1 week prior to experiments in a temperature-controlled room (25±2°C) with 12-hour light and dark cycles. Animals were randomly divided into 3 groups: control (sham burn), burn, and burn plus ML-9 treatment group.ad libitum, and recovered from anesthesia. At the end of experiment, blood of the anesthetized mice from each experimental group was collected for intestinal permeability assay. Then, mice were sacrificed for tissue harvest. The harvested tissues were used for histological, immunofluorescent, and biochemical evaluation, as described blow.After acclimatization, mice assigned to burn group received a 30% total body surface area (TBSA) full thickness burn. Briefly, following a 12-h fast with water available ad libitum, animals were anesthetized with an intraperitoneal injection of sodium pentobarbital (30 mg/kg). Under anesthesia, the dorsal fur of the animals was shaved and depilated with 8% sodium sulfide. A 30% TBSA full-thickness flame burn was produced by igniting 3% napalm for 10 seconds on the depilated dorsum. Mice in control group received treatment similar to burn animals with the exception of the flame burn treatment, whereas the mice assigned to burn plus ML-9 treatment group were received an immediately intraperitoneal injection of ML-9  dissolved in saline after flame burn treatment. After injury, mice were immediately resuscitated with an intraperitoneal injection of 2.0 ml lactated Ringer's solution. Then, mice were individually caged, provided food and water Intestinal paracellular permeability was determined by measuring the appearance in blood of a marker, 4.4 kDa fluorescein isothiocyanate-labeled dextran (FITC-dextran) . The assay of intestinal paracellular permeability was slightly modified from the previously described methods The ileal tissues were promptly rinsed with cold 0.9% saline solution and immediately fixed in 10% buffered formalin phosphate (pH 7.0) until processing for histological sections. The formalin-fixed samples were embedded in paraffin, and sectioned. After deparaffinization and dehydration, the sections were stained with hematoxylin and eosin for histological assessment of intestinal mucosa. Histological changes of intestinal mucosa were observed with a DM6000B microscope , and images were obtained using Metamorph 7.5 software .2 for 30 min at room temperature, then washed thrice with PBS for 5 min, permeabilized in 1% Triton X-100 in PBS at room temperature for 5 min. After washing with PBS, the nonspecific binding sites were blocked with 5% normal goat serum in PBS for 30 min. Then, the sections were incubated with monoclonal rabbit antibody against ZO-1, occludin or claudin-1  diluted at 1∶200 with 1% normal goat serum in PBS at 4°C overnight. After washing thrice in PBS, sections were incubated with secondary Alexa Fluor 488 conjugated goat anti-rabbit IgG antibody (Invitrogen) at 1∶100, Alexa Fluor 594-conjugated phalloidin (Invitrogen) at 5 U/ml, and DAPI (Sigma) for 1 hour at room temperature. After washing thrice in PBS, sections were mounted using Slowfade reagents (Invitrogen). Images were obtained using a TCS SP5 laser confocal microscopy .Frozen sections of ileal tissue were fixed with 1% paraformaldehyde in PBS containing 1 mmol/L CaClAfter the animals were sacrificed, a 5 cm ileal segment was taken to harvest mucosa by a glass slide. The harvested mucosa were homogenized with 10 volumes of ice-cold RIPA buffer containing 150 mM NaCl, 10 mM Tris-HCl, pH 7.5, 1% sodium deoxycholate, 1% NP-40, 10 mM EDTA, 0.1% SDS, including both protease and phosphatase inhibitor cocktail (Sigma), followed by a brief sonication with a sonicator . Thereafter, the homogenates were centrifuged at 4°C, 15000 g for 10 min, and the supernatants were collected to determine protein concentration using the RC DC protein assay kit . For the determination of MLCK, MLC and pMLC, equal amounts of total protein extracted from the ileal mucosa were fractionated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and then transferred to polyvinylidene difluoride (PVDF) membrane . After complete transfer, membranes were blocked for 60 min at room temperature with 5% nonfat milk dissolved in TBST buffer. After blocking, membranes were incubated with antibodies specific for MLCK , MLC , phosphorylated MLC , and β-actin  overnight at 4°C. After wash with TBST, membranes were incubated with appropriate peroxidase-conjugated secondary antibodies  at room temperature for 60 min. The blots were visualized using Super Signal West Pico reagent . The chemiluminescence signal was captured using a ChemiDoc XRS system (Bio-Rad). The densities of the bands were quantified with Quantity One Image software (Bio-Rad).p value of <0.05 was considered as the minimum level of significance in all cases. All reported significance levels represent two-tailed p values.Data are presented as means ± SEM. For multi-group data analysis, one-way analysis of variance (ANOVA) was performed using SPSS 13.0 statistical software. A in vivo, we first did the time-course analysis of intestinal paracellular permeability in mice subjected to 30% TBSA full-thickness burn. As shown in By measuring the appearance in blood of 4.4 kDa FITC-dextran which was traditionally used to measure the movement of small molecules across the intestinal epithelium in vivo and in vitroin vivo inhibiting MLC phosphorylation with ML-9, an MLCK inhibitor, could ameliorate the increased intestinal permeability induced by severe burn injury, and selected postburn 6 h as the time point for further studies. Our preliminary experiments proved that ML-9 alone had no obvious effect on intestinal permeability in normal balb/c mice (data not shown). As illustrated in in vivo inhibition of MLCK activity is capable of attenuating the increased intestinal permeability induced by severe burn injury.It has been well recognized that increased MLC phosphorylation triggered by activated MLCK is critical to intestinal barrier defect both in vivo inhibition of MLCK activity with specific inhibitor attenuates the intestinal mucosa injury following severe burns.As shown in To more precisely understand the intestinal barrier defect induced by burn injury, we next evaluated the morphological changes of zonula occludens-1 (ZO-1), a member of tight junction proteins, by immunofluorescent antibody labeling assay. As shown in Tight junction protein occludin was mainly localized in cytoplasm and epithelial tight junctions in the ileum of control mice . After bIt has been well documented that MLCK-dependent MLC phosphorylation is critical to intestinal barrier disruption MLCK has been proven to be the predominant determinant of MLC phosphorylation in intestinal epithelial cells in vivo. These findings provide a new insight into the mechanisms involved in the intestinal barrier breakdown caused by severe burn injury.In this study, it is demonstrated that severe burn injury causes the increase of intestinal permeability, and that the increased intestinal permeability induced by burn injury is accompanied by the histological damage of intestinal mucosa, redistribution of tight junction protein ZO-1 as well as upregulation of both MLCK protein and MLC phosphorylation. Our present study also reveals that MLCK inhibition with specific inhibitor ML-9 attenuates the burn-induced intestinal barrier disruption It has been well documented that many critical surgical diseases, such as shock, trauma and burn injury, causes the disruption of intestinal epithelial barrier function, leading to the leakage of bacteria, microbial products or other antigens from the intestinal lumen into the mucosa or systemic circulation to initiate or exacerbate an inflammatory response Given that the intestinal barrier disruption is induced following severe burn, the underlying mechanisms are still incompletely elucidated. Actually, the early systemic and intestinal damage caused by thermal injury is orchestrated by a series of pathophysiological events such as ischemia, hypoxia, and inflammation, which have been demonstrated to contribute to intestinal barrier dysfunction. We and others have previously shown that hypoxia or ischemia, which occurs rapidly following severe burns, is capable of causing barrier disruption Intestinal barrier function disruption is characterized by the increased paracellular permeability as well as the changes of tight junction protein expression and organization in vitro studies have shown that increased MLC phosphorylation mediated by upregulated MLCK protein expression is critical to barrier breakdown induced by hypoxia or proinflammatory cytokines MLCK activation, which directly leads to the phosphorylation of MLC, has been viewed as a common final pathway of acute tight junction regulation in response to a broad range of stimuli It should be noted that MLCK inhibition with ML-9 just ameliorates, but not corrects, the burn-caused intestinal barrier breakdown. Thus, other signaling pathways should be considered in the pathogenesis of intestinal barrier disruption following severe burn injury. For example, the accumulating published data have shown that the percentage of apoptotic intestinal epithelial cells is significantly increased in mice suffering from 30% TBSA cutaneous full-thickness burn in vivo experimental study providing direct evidence to show the role of MLCK-dependent MLC phosphorylation in burn-induced intestinal epithelial barrier disruption. It is suggested that MLCK-dependent MLC phosphorylation may be a critical target for the therapeutic treatment of intestinal epithelial barrier disruption after severe burn injury.In summary, our present data demonstrate the involvement of MLCK-dependent MLC phosphorylation signaling pathway in intestinal barrier dysfunction following severe burn injury. To the best of our knowledge, this is the first


We aimed to investigate sensitization profiles of egg allergic patients and compare Egg allergic children (n = 25) were subjected to skin prick test, double blind placebo controlled food challenge, and sensitization profiles to Gal d 1–5 were determined by allergen microarray. IgE binding and biological activity of eggs from different chicken breeds were investigated by immunoblot, ELISA, and mediator release assays.We found that Gal d 1 and Gal d 2 are generally major egg allergens, whereas Gal d 3–5 displayed high sensitization prevalence only in patients reacting to both, egg white and yolk. It seems that the onset of egg allergy is mediated by egg white allergens expanding to yolk sensitization in later stages of disease. Of note, egg white/yolk weight ratios were reduced in eggs from Auraucana and Maran chicken. As determined in IgE immunoblots and mass analysis, eggs from ancient chicken breeds did not differ in their protein composition. Similar IgE-binding was observed for all egg white preparations, while an elevated allergenicity was detected in egg yolk from Araucana chicken.Our results on allergenicity and biological activity do not confirm the common assumption that aboriginal food might be less allergenic. Comprehensive diagnosis of egg allergy should distinguish between reactivity to hen's egg white and yolk fractions to avoid unnecessary dietary restrictions to improve life quality of the allergic child and its family. Approximately 15% of the overall population suffers from IgE-mediated adverse reactions upon the ingestion of several kinds of food. According to the sensitization process, this disease can be divided into primary and secondary food allergies, affecting 9.4% and 5.5% of the population, respectively Gallus domesticus) allergens while nine more IgE-binding egg proteins are listed in the Allergome database . Ovomucoid , ovalbumin , ovotransferrin , and lysozyme  constitute the most abundant proteins in the egg white fraction and have all been described as major allergens Allergic reactions to hen's egg represent one of the most frequent primary food allergies affecting around 1.6% of children below the age of three, but are in the majority of cases outgrown before school age Although to a lower frequency, allergic reactions to yolk have been reported in vitro and clinical allergy diagnosis.Egg allergy has a significant effect on the quality of life and although symptoms are usually not life-threatening, severe anaphylactic reactions upon egg consumption have been reported P = 0.015) in the group of patients displaying additional sensitization to yolk (median 3.56 µg/ml versus 1.02 µg/ml). With 6.8±3.6 years (mean ± STD), the average age in this group was also slightly higher than for children belonging to the yolk-negative group (4.3±2.3 years). Moreover, the two groups differed in their allergen sensitization pattern , even though those proteins are considered egg white allergens. As expected, IgE reactivity to Gal d 5 was exclusively detected in yolk-sensitized patients.Studying a large cohort of 474 Italian subjects  pattern . In micrAs illustrated in P<0.001) constituting 1.7 and 2.4 for ancient chicken breeds and modern laying hens, respectively.Weights and pH values of eggs from Araucana, Maran, and laying hen hybrid chicken were determined in the laboratory . No signEgg white and yolk extracts from Araucana, Maran, and modern laying hen hybrids displayed similar protein composition and IgE-reactivity patterns as revealed by gel electrophoresis and IgE immunoblot analysis . Mass-spELISA experiments showed that thermal denaturation had only a minor impact on the IgE reactivity of egg extracts . No signELISA experiments revealed similar levels of IgE reactivity of eggs from different breeds . Mean eget al, unpublished). On the molecular level, our and recent data e.g. the recently identified Gal d 6 Although in the majority of cases outgrown before school age, egg allergy can drastically decrease life quality due to the omnipresence of egg containing food products. Avoidance is usually difficult and implies broad dietary restrictions Although eggs from different birds contain cross-reactive allergens, the development of egg allergy seems to be restricted to distinct avian species in vitro data showing no advantage of eggs from ancient breeds, food challenges with Auraucana and Maran eggs were considered of minor scientific benefit. Moreover, it has previously been shown that continuous egg intake boosts systemic IgE responses in allergic patients i.e. higher cholesterol and lower protein content, have been previously shown We did not find major dissimilarities regarding IgE reactivity or biological activity between eggs from modern and ancient chicken breeds. Noteworthy, egg yolk from Auraucana chicken even showed a slightly elevated allergenic potential. Based on these clear per se contributes little to primary egg allergy. By contrast, it seems that the egg, and in particular sensitization to egg white proteins, comes first. A picture is emerging in which the development of egg allergy seems to start with sensitization to Gal d 1 and Gal d 2. In the course of disease children raise additional IgE antibodies against other egg white  and finally yolk allergens. This hypothesis is supported by our allergen profiling data and the fact that yolk-negative patients were by trend younger. Although additional yolk sensitization does not seem to cause more severe symptoms, it may worsen life quality because it enlarges dietary restrictions.So which then came first, the chicken or the egg? From the allergological point of view this intriguing question might be answered as follows: Although representing a putative sensitizer in the “bird-egg syndrome” In summary, eggs from ancient chicken breeds cannot be considered less allergenic but might be better tolerated by some not severely affected patients due to their smaller egg white fraction. There are strong variations regarding the number and type of egg-containing foods that an individual patient can eat without experiencing adverse reactions. Therefore, detailed and molecule-based diagnosis distinguishing between sensitization to egg white and yolk is essential for improved patient management.The study was approved by the Institutional Review Board of Istituto Dermopatico dell'Immacolata – IDI-IRCCS, Rome, Italy (n. 106-CE-2005), and signed informed consents were obtained from legal guardians.Sera were obtained from the Center for Molecular Allergology at IDI-IRCCS in Rome, Italy. Egg allergic children were selected according to typical case history and positive skin reactivity. Skin prick tests (SPT) were performed as previously reported Children were admitted to the Clinical Center in a fasting state. Administration of antihistamines, if used, was stopped at least 7 days before provocation test and none of the patients was under steroid treatment. Food challenge was carried out with commercially available raw eggs from hybrid hens in two different days. Eggs were mixed with 50 ml of non-sparkling mineral water plus two teaspoons of cocoa  and one spoon of white sugar (verum). After administration of an initial dose of 0.1 ml, amounts were increased to 1 ml and 10 ml in 30 minute intervals. Residual egg preparations were given until objective symptoms developed or until the equivalent of one egg was ingested.i.e. Araucana and Maran, were obtained from the Hölzl poultry hatchery in Moosburg, Germany. Medium-sized eggs from conventional laying hen hybrids were purchased at the local grocery. Average weights from whole egg, egg white, and yolk, as well as egg white to yolk weight ratios were determined for 10 individual eggs from each chicken breed. Moreover, pH values of egg white and yolk fractions were measured with Whatman pH indicator stripes. For preparations of allergen extracts, 3 ml samples of yolk and egg white fractions were taken from 10 individual cracked eggs. To avoid egg white contaminations, yolks were gently punctured with a syringe and samples were taken carefully. Pooled samples were extracted overnight at 4°C in PBS pH 7.4 containing 2% polyvinylpyrrolidone, 2 mM phenylmethanesulfonylfluoride, 2 mM benzamidine, and 2 mM ethylenediaminetetraacetic acid at a concentration of 1 g/ml. After centrifugation at 21,000 g and 4°C for 30 min, samples were sonicated, and filtered through a 0.45 µm filter unit . Protein concentrations were determined by the method of Bradford, and egg extracts were stored at 4°C.Eggs from ancient chicken breeds, 125I-labelled anti human IgE . Radiographic signals were visualized by overnight exposure to imaging plates and subsequent evaluation using a BAS-1800II Phosphor Imager scanner .Egg white and yolk extracts were separated by SDS-PAGE under reducing conditions and stained with GelCode® Blue Reagent . For immunoblots, extracts were separated by gel electrophoresis and electroblotted onto nitrocellulose Protan membranes . After blocking unspecific binding sites with 25 mM Tris HCl, pH 7.5, 0.5% bovine serum albumin (w/v), 150 mM NaCl, and 0.05% Tween 20 (v/v), membranes were incubated with a serum pool (n = 25) diluted 1∶10 in the blocking buffer. Bound IgE was detected using IgE-reactive bands were excised from polyacrylamide gels and subjected to in gel digestion using the ProteoExtract All-In-One Trypsin Digestion Kit . Tryptic peptides were separated by capillary reversed phase high pressure liquid chromatography (rpHPLC) directly coupled to a Quadrupole-Time of Flight mass spectrometer  equipped with a Nanoflow spray head. Obtained mass data were processed and analyzed using the software Protein Lynx Global Server version 2.2.5  and evaluated using the SwissProt/TrEMBL database.Maxisorp plates  were coated with 250 ng of egg extracts in PBS pH 7.4 overnight at 4°C. To assess IgE reactivity to heat-denatured egg allergens, egg extracts were incubated for 15 minutes at 95°C before use. After blocking unspecific binding sites with Tris-buffered saline pH 7.4, 1% bovine serum albumin (w/v), and 0.05% Tween 20 (v/v), coated antigen was incubated with patient's sera overnight at 4°C. Bound IgE was detected with an alkaline phosphatase-conjugated monoclonal anti human IgE antibody . Specific serum IgE levels were calculated based on a semi-quantitative ELISA using purified human IgE  as internal standard.Rat basophil degranulation assays were performed as previously described t-test were used for non-paired samples. A value of P<0.02 was considered statistically significant.Statistical evaluation of paired samples was performed using the Wilcoxon signed rank test; Mann-Whitney rank sum test and


This complex binds to the Cdc25A DSG motif which contains serine residues at positions 82 and 88. Phosphorylation of one or both residues is necessary for the binding and degradation to occur.Failure to regulate the levels of Cdc25A phosphatase during the cell cycle or during a checkpoint response causes bypass of DNA damage and replication checkpoints resulting in genomic instability and cancer. During G1 and S and in cellular response to DNA damage, Cdc25A is targeted for degradation through the Skp1-cullin-β-TrCP , however, and therefore does not suppress ASK1-mediated apoptosis.We now show that mutation of serine 88 to phenylalanine, which is a cancer-predisposing polymorphic variant in humans, leads to early embryonic lethality in mice. The mutant protein retains its phosphatase activity both These data suggest that the DSG motif, in addition to its function in Cdc25A-mediated degradation, plays a role in cell survival during early embyogenesis through suppression of ASK1-mediated apoptosis. Cip through activation of p53 [When DNA is damaged or when blocks to DNA replication occur, cells activate an extensive network of signaling pathways that promote cell cycle arrest and DNA repair . Cell cyn of p53 . The secβ-TrCP) complex and the anaphase-promoting complex (APCCdh1), participate in Cdc25A turnover. The APC/C proteasome complex helps regulate Cdc25A at the exit of mitosis while SCFβ-TrCP regulates the abundance of Cdc25A in S phase and G2 [Cdc25A phosphatase is an essential activator of cell cycle progression and its expression is tightly regulated at many levels, including transcriptional activation, reversible phosphorylation, protein-protein interaction and ubiquitin-mediated degradation -9. Ubique and G2 ,10-13. We and G2 ,15. Overe and G2 , leads te and G2  and mitoβ-TrCP-mediated degradation [C. elegans at a serine equivalent to human serine 88 is a gain of function mutation that causes deregulated intestinal cell hyper-proliferation and hyperplasia [The ubiquitinylation-mediated degradation of Cdc25A is associated with phosphorylation of Cdc25A at two residues within the β-TrCP docking site (DSG motif). The DSG docking site of Cdc25A is comprised of serines 79, 82 and 88, and the absence of their phosphorylation is sufficient to abolish β-TrCP binding and interfere with Cdc25A degradation . Severalradation -20. The radation . The exiradation  that eleradation  underscoerplasia .β-TrCP-mediated degradation. Inhibition of Cdc25A degradation in turn should lead to its accumulation, accompanied by bypass of DNA damage and replication checkpoints, enhanced DNA damage and increased risk of cancer. In the mouse, we find that this mutation, when homozygous, produces early fetal lethality, indicating that the Cdc25A DSG motif and serine 88 phosphorylation may also have a role in early embryogenesis. Homozygous mutant embryos that persist display an altered morphology with extensive cellular degeneration and die at or prior to embryonic day 3.5. Circular dichroism (CD) studies revealed that the S88F substitution in Cdc25A protein had no major effect on protein secondary structure that would account for the observed phenotype. The mutant protein retains phosphatase activity but, unlike the wildtype Cdc25A, the S88F mutant fails to interact with the apoptosis signal-regulating kinase-1 (ASK1) facilitating apoptosis mediated by ASK1. These data demonstrate that the DSG domain of Cdc25A has a role in cell survival during early embryogenesis that is separate from its role in Cdc25A ubiquitin-mediated degradation in response to DNA damage.To better understand the significance of Cdc25A phosphorylation within the DSG domain in cell cycle regulation and DNA damage response, we generated a knock-in mouse model with a substitution of phenylalanine for serine at residue 88. The expectation was that cells from mice harboring this Cdc25A variant would be less susceptible to SCFβ-TrCP-mediated degradation following etoposide treatment. Substitution of either serine 82 or 88 to alanine individually was sufficient to confer partial resistance to degradation of Cdc25A following DNA damage . The CD spectra of the two proteins were similar, although they revealed small but significant differences Figure . The simin vitro and in cultured cells. To measure relative phosphatase activities in vitro, the activities of wildtype Cdc25A, Cdc25A S88F and a Cdc25A S88A mutant were assayed by hydrolysis of 4-nitrophenol phosphate (pNPP). The phosphatase activity of Cdc25A S88F mutant is similar to that of wildtype Cdc25A and Cdc25A S88A, indicating that the serine to phenylalanine substitution does not inactivate Cdc25A phosphatase activity in vitro  in which serine 88, one of two key serine residues in the DSG domain, is substituted with a phenylalanine. This serine residue is important for β-TrCP binding and for ubiquitin-dependent degradation of Cdc25A protein. The expectation was that mice expressing Cdc25A S88F would have increased amounts of the protein due to a reduction in its degradation and that cells would undergo unscheduled replication, display genomic instability and early tumor onset in the progeny. No mice that were homozygous for Cdc25A S88F, however, were born. The Cdc25A is a transcriptional target of c-Myc and that overabundance of Cdc25A suppresses apoptotic cell death [Since mutant Cdc25A displays no significant change in protein conformation or phosphatase activity, it is most likely that the lethal phenotype is due to an alternative Cdc25A function. It is known that ll death ,27. One ll death . When Cdll death . We therll death . Failureneo marker but with a single remaining intronic loxP site. All animal experiments were carried out under protocols approved by the Institutional Animal Care and Use Committee of the University of Cincinnati.Two fragments of Cdc25A genomic DNA were PCR amplified and cloned in a pBSK-TK vector. The first fragment (5030 bp) contained a point mutation GATTC to GATTT (on exon 3) at position 4780. A neomycin resistance marker flanked by loxP sites (LoxP-PGK-Neo-LoxP) was included in the intron 3 immediately after the mutation. A second homologous arm (6715 bp) was cloned after the Neo cassette using conventional molecular biology techniques. The sequence of the targeting construct was validated by direct sequencing. The vector was linearized at a unique Not I site and electroporated into 129/Sv ES cells and neomycin-resistant colonies were screened for correct targeting by Southern blots. Two faithfully targeted ES cell lines were identified, and both were injected into the blastocoel of 3.5-day-old blastocysts which were implanted into the uterus of recipient mice. Chimeras carrying the targeted allele were identified, mated with Black Swiss mice, and agouti progeny were assayed for transmission of the knock-in allele. Mice that were homozygous for the S88F variant were crossed with TgN(EIIa-Cre)C79Lmgd mice  in an FVB/N strain background. Cre recombinase is selectively expressed in the germline of the progeny mice so that their offspring will retain the targeted allele without the -1cm-1, as determined by Sednterp [C is the concentration in molar units, l is the pathlength in cm, and nr is the number of residues. Data were analyzed using CDSSTR [GST-tagged Cdc25A-wt and Cdc25A S88F proteins were purified using a combination of ammonium sulfate precipitation (65% cut-off), a size exclusion column fractionation, a sulphopropyl (SP) column to separate the GST tag from Cdc25A protein following GST tag removal from the fusion protein using PreScission™ Protease (Sigma), and a glutathione affinity column run according to the manufacturer's protocol (Sigma). Purified wildtype and mutant Cdc25A far-UV CD spectra were measured using an Aviv 215 circular dichroism spectrometer. Proteins were dialyzed into 10 mM Tris pH 8.0, 150 mM NaCl, 1% ethylene glycol, and 2 mM β-mercaptoethanol, and spectra were measured from 260 to 190 nm. Protein concentrations were determined using the absorbance at 280 nm and a molar extinction coefficient of 31,860 MSednterp . Data weg CDSSTR  in Dichrp-nitrophenolate at 410 nm and subtracting the control background value. Each point is the mean ± SEM of data from two separate experiments.The phosphatase activity of Cdc25A was assayed by hydrolysis of 4-nitrophenol phosphate (pNPP)  as described previously  with mod2). Cells (2 × 105) were incubated with 5 μl Annexin V-Cy5.5 and PI (1 mg/ml) for 10 min in the dark at room temperature and analyzed by flow cytometry.Apoptosis was measured using Annexin V-Cy5.5  and propidium iodide (PI). Briefly, harvested cells were washed in phosphate-buffered saline (PBS) and resuspended in Annexin V binding buffer . Fluorescent green cells were then visualized with the use of a LSM510 laser scanning confocal or Orca microscope .The authors declare that they have no competing interests.EMB planned and performed the experiments, analyzed the data and wrote the paper. MY, YQL, SBR, DGC and ZY, performed the experiments, RAK, ABH planned and analyzed the data and PJS planned and analyzed the data and wrote the paper. All the authors read and approved the final manuscript.


O-β-glucoside (Cy-3-g), a typical anthocyanin pigment was used to examine its effects on AMPK activation and fatty acid metabolism in human HepG2 hepatocytes.Hepatic metabolic derangements are key components in the development of fatty liver disease. AMP-activated protein kinase (AMPK) plays a central role in controlling hepatic lipid metabolism through modulating the downstream acetyl CoA carboxylase (ACC) and carnitine palmitoyl transferase 1 (CPT-1) pathway. In this study, cyanidin-3-Anthocyanin Cy-3-g increased cellular AMPK activity in a calmodulin kinase kinase dependent manner. Furthermore, Cy-3-g substantially induced AMPK downstream target ACC phosphorylation and inactivation, and then decreased malonyl CoA contents, leading to stimulation of CPT-1 expression and significant increase of fatty acid oxidation in HepG2 cells. These effects of Cy-3-g are largely abolished by pharmacological and genetic inhibition of AMPK.This study demonstrates that Cy-3-g regulates hepatic lipid homeostasis via an AMPK-dependent signaling pathway. Targeting AMPK activation by anthocyanin may represent a promising approach for the prevention and treatment of obesity-related nonalcoholic fatty liver disease. Nonalcoholic fatty liver disease (NAFLD) is a serious consequence of obesity, increasing the risk of liver cancer or cirrhosis . The oriAMP-activated protein kinase (AMPK) is a key sensor of cellular energy status and it is also recognized as a major regulator of liver and whole body lipid homeostasis . AMPK acAnthocyanins are naturally occurring polyphenolic compounds in the plant foods and widely distributed in fruits, vegetables, and pigmented cereals, suggesting that we can ingest significant amounts of anthocyanins from plant-based daily diets . ClinicaO-β-glucoside (Cy-3-g), the most abundant anthocyanin in plants i), without detectable changes in the AMP/ATP ratio [2+ signaling was involved in the AMPK activation upon exposure of HepG2 cells to anthocyanin. Anthocyanin exposure resulted in a rapid and transient increase in [Ca2+]i concentration  triggered by a rise in intracellular calcium ions  was infected into HepG2 cells. The lack of AMPK significantly compromised the anthocyanin-mediated reduction of malonyl CoA levels Figure . As a coThis study demonstrates that Cy-3-g, a typical anthocyanin pigment, stimulates AMPK activation via CaMKK in HepG2 cells. The increase in AMPK activation by anthocyanin suppresses ACC activity, causes the reduction in malonyl CoA levels and stimulation of CPT-1, leading to enhanced fatty acid β-oxidation and finally inhibiting the lipid accumulation in HepG2 cells. Thus, the novel findings from the present study is that anthocyanin beneficially regulates hepatic fatty acid metabolism through AMPK-ACC-malonyl CoA-CPT1 pathway.Previous studies had shown that Cy-3-g activated AMPK and enhanced adipocytokine secretion and adipocyte-specific gene expression in isolated adipocytes ,19. Rece2+. We found that exposing HepG2 cells to anthocyanin initiated a rapid and transient influx of Ca2+ into the cells and that AMPK activation by anthocyanin was prevented by the intracellular Ca2+-chelating agent, BAPTA-AM, suggesting that the anthocyanin-induced signaling pathway was dependent upon intracellular Ca2+. Since CaMKK-β-mediated stimulation of AMPK required an increase in intracellular Ca2+ levels, we hypothesized that CaMKK-β may be the upstream kinase induced by anthocyanin. Indeed, the CaMKK inhibitor STO-609 decreased the baseline AMPK activity and prevented activation of the AMPK upon anthocyanin exposure. Moreover, when transfected with CaMKK-β siRNA, both baseline and anthocyanin-induced activity of AMPK were inhibited. These data suggest that the anthocyanin-induced activation of AMPK was mediated by CaMKK-β independently of LKB1 signaling pathway.In mammals, LKB1 and CaMKK-β have been identified as upstream kinases of AMPK, since both kinases can phosphorylate Thr172 and thus activate AMPK ,21. The In the liver, AMPK coordinates the changes in the activity of enzymes of lipid metabolism and regulates the partitioning of fatty acid both in oxidative and biosynthetic pathways . In accoIn summary, our present study provide the mechanistic data that activation of hepatic AMPK by anthocyanin acts upstream on lipid metabolism by activating the pathways controlling the oxidation of fatty acids. Although it appears hard to get the concentration of anthocyanin shown in this study from daily consumed foods, our findings provide a novel insight into the therapeutic implications of anthocyanin in obesity-related liver disorders.O-β-glucoside  was provided by Polyphenol AS . Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and antibiotic mixture (penicillins-treptomycin) were purchased from the Gibco BRL . 5-aminoimidazole-4-carboxamide 1-ribofuranoside (AICAR), anti-phospho-AMPK Thr-172, anti-phospho-ACC Ser-79 and anti-β-actin were purchased from Cell Signaling Technology, Inc. . AMPK inhibitor compound C and calmodulin-dependent kinase kinase (CaMKK) inhibitor STO-609 were obtained from Calbiochem . The specific AMPK-targeted SAMS peptide used in AMPK activity assay was purchased from GenScript ; siRNA against CaMKK-β was from Qiagen, Inc. ; and all other chemicals, unless otherwise specified, from Sigma-Aldrich .Anthocyanin cyanidin-3-2/95% air atmosphere. After reaching ~80% confluence, cells were serum-starved for 24 h in medium containing 0.5% FBS.HepG2 cells  were cultured in DMEM supplemented with 10% FBS and antibiotics at 37°C in a humidified, 5% COThe adenoviral vector expressing a dominant-negative AMPKα mutant (Ad-DN-AMPK) was kindly donated by Dr J. Ha .AMPK activity was measured by monitoring phosphorylation of the SAMS peptide substrate as previously described . To dete2HPO4 (pH 7.2), 2 mM MgSO4, 1 mM β-NADP (β-nicotinamide adenine dinucleotide phosphate), 1.4 U/mL G-6-PDH (glucose-6-phosphate-dehydrogenase) and 3 U/mL PGM (phosphoglucomutase). AMP or Cy-3-g was added to the assay medium at the specified concentrations followed by the addition of glycogen  to initiate the reaction. After incubating 10 min at 25°C, GPPase activity was assessed by measuring absorbance at 340 nm i). Cells were incubated at 25°C for 60 min with 1 μM fura-2-AM in modified Krebs Ringer buffer . After dye loading, cells were washed three times in the same medium and kept in the dark for at least 30 min before single-cell [Ca2+]i measurements. Changes in [Ca2+]i were determined ratiometrically  in 1-mL aliquots using a spectrofluorometer. Calcium concentrations were calculated using the equation: [Ca2+]i = Kd(F380 max/F380 min) (R - Rmin)/(Rmax - R) acetyl-CoA, and purified fatty acid synthetase (FAS) was measured using a modification of a previously described method acetate  and AICAR or Cy-3-g at the indicated concentrations. Cells were incubated 4 h at 37°C then the plates were rinsed with PBS. The final wash was replaced with Microscint20 (Perkin Elmer) and radioactivity incorporated into fatty acid monitored on MicroBeta Liquid Scintillation and Luminescence Counters.HepG2 cells  containing complete protease and phosphatase inhibitors , and then immunoblotted with antibodies against p-AMPK, AMPK, and p-ACC, ACC for 2 h. Specific antibody binding was detected by horseradish peroxidase-conjugated secondary antibodies and visualized using enhanced chemiluminescence detection reagent (Santa Cruz). The band densities were quantified using an image analyzer Quantity One System . All protein quantifications were adjusted for the corresponding β-actin level, which was not consistently changed by the different treatment conditions.t test or analysis of variance (ANOVA) to determine P values; P < 0.05 was considered statistically significant.Statistical analyses were performed using the SPSS 14.0 package . All results are expressed as means ± SE. and analyzed by the Student's ACC: acetyl-CoA carboxylase; AMPK: AMP-activated protein kinase; CaMKK: calmodulin-dependent protein kinase kinase; CPT1: carnitine palmitoyl-CoA transferase 1; Cy-3-g: cyanidin-3-O-β-glucoside; FAS: fatty acid synthase; FBPase: fructose-1,6 diphosphatase; GPPase: glycogen phosphorylase; MCD: malonyl CoA decarboxylase; NAFLD: nonalcoholic fatty liver disease; TG: triglycerideThe authors declare that they have no competing interests.HG and MX conceived the idea and designed the study. DW and GL carried out the lipid metabolism assay experiments. RZ performed the statistical analysis and interpretation of the data. YW participated in the experiments related to AMPK enzyme activity assay. HG drafted the manuscript and MX provided critical corrections to the manuscript. All authors read and approved the final manuscript.


This carbohydrate specificity is mediated by the type 1 fimbrial lectin FimH. In this account, we have employed four different set-ups to assay type 1 fimbriae-mediated bacterial adhesion, including tailor-made glycoarrays. The focus of our study was on testing FimH specificity with regard to the glycone part of a glycosidic ligand by testing a series of synthetic α-mannosides, as well as α-glucosides and α-galactosides. Unexpectedly, it was found that in solution all tested aminothiahexyl glycosides inhibit bacterial adhesion but that this effect is unspecific. Instead it is due to cytotoxicity of the respective glycosides at high mm concentrations.Adhesion of bacteria to the glycosylated surface of their target cells is typically mediated by fimbrial lectins, exposed on the bacterial surface. Among the best-investigated and most important fimbriae are type 1 fimbriae, for which α- Escherichia coli is a widely distributed bacterial species that is responsible for many serious infections. Urogenital infections, for example, are caused by uropathogenic E. coli (UPEC). In order to infect their target cells, UPEC first have to accomplish adhesion to the glycosylated cell surface and establish colonization of the cell surface [E. coli and constitute important virulence factors of uropathogenic strains. They are used to mediate attachment to specific niches in the urinary tract [ surface ,2. The b surface ,4. Type ry tract . Thus, try tract ,7.N-linked high-mannose type oligosaccharides serve as receptor molecules for type 1 fimbriae. Early studies by Sharon and Ofek et al. have revealed the affinities of various oligosaccharides of different complexity [d-mannosyl moieties, which varies in different oligosaccharides, is important for binding to type 1-fimbriated bacteria. This assumption is also supported by recent literature on carbohydrate binding of selectins [It has been shown that glycoproteins carrying one or more mplexity ,10,11,12electins . Additioelectins ,22,23,24electins ,28,29,30electins ,32.P anchoring the lectin at the tip of the type 1 fimbrial shaft and its lectin domain, FimHL, harboring the carbohydrate-binding site. X-ray analysis of FimH has shown that [d-mannosyl residue can be accommodated within the carbohydrate-binding pocket (β-glycosides do not fit into the binding site). The aglycone moiety of a natural oligosaccharide exerts additional interactions at the periphery of the carbohydrate-binding site [d-mannoside according to computer docking and biological testing [d-mannosides more strongly [Apparently, carbohydrate binding of type 1 fimbriae is mediated by the lectin FimH, which is located at the fimbrial tips . FimH isown that ,35,36,37 testing ,38. This testing ,40,41,42 testing . Tensilestrongly .manno-, gluco-, and galacto-series, 1–6  A binding assay with GFP-tagged E. coli to microtiter plate-based glycoarrays testing varied bacterial concentration and (ii) varied glycoarray density; (iii) an adhesion-inhibition assay to test the prepared series of synthetic α-glycosides as inhibitors of bacterial adhesion to a mannan-coated surface in solution, and (iv) a preincubation-inhibition-adhesion assay in which the bacteria are allowed to interact with the glycosides in solution before they are transferred to the microplates. This approach should allow us to test if in any set-up an altered carbohydrate specificity of type 1 fimbriae-mediated bacterial adhesion can be seen.Carbohydrate specificity of lectins is typically tested in solution or on polystyrene microtiter plates, which can be carbohydrate-decorated to allow type 1 fimbriae-mediated adhesion of E. coli . The pot1–6) were synthesized according to known procedures. The aminophenyl glycosides, 1, 3, and 5, were obtained by catalytic reduction from the corresponding nitrophenyl glycosides, which are commercially available. The 6-amino-4-thiahexyl glycosides, 2, 4, and 6, where synthesized starting with the respective allyl glycosides by radical addition of cysteamine [All glycosides used for the bacterial adhesion assays , 1–6 wersteamine . All pre®20 (0.05% v/v). Lysogeny broth (LB) medium: tryptone (10.0 g), sodium chloride (10.0 g), and yeast extract (5.00 g) were dissolved in distilled deionized water (1.00 L) and autoclaved, and ampicillin (100 mg) and chloramphenicol (50 mg) were added. Agar plates: tryptone (10.0 g), sodium chloride (10.0 g), yeast extract (5.00 g), and agar (15.0 g) were dissolved in distilled deionized water (1.00 L) and autoclaved, and ampicillin (100 mg) and chloramphenicol (50 mg) were added. All pH values were adjusted with HCl (1 m) and NaOH (1 m).Carbonate buffer solution (pH 9.2): sodium carbonate (1.59 g) and sodium hydrogen carbonate (2.52 g) were dissolved in distilled deionized water. PBS buffer solution (pH 7.2): sodium chloride (8.00 g), potassium chloride (200 mg), sodium hydrogen phosphate dihydrate (1.44 g), and potassium dihydrogen phosphate (200 mg) were dissolved in distilled deionized water (1.00 L). PBST buffer solution: PBS buffer + TweenE. coli strain pPKL1162 was used [E. coli mutant exclusively expresses type 1 fimbriae as well as GFP (green fluorescent protein) allowing for quantification of bacterial adhesion by fluorescence read-out [E. coli strain pPKL1162 was constructed by inserting the pPKL1174 plasmid into strain SAR18. The pPKL1174 plasmid contains the fim gene cluster, which is responsible for the expression of type 1 fimbriae. SAR18 includes the GFP gene on its genome, controlled by a constitutive promoter [600 = 0.4 (≙ 2 mg/mL) with PBS.For testing the GFP-expressing was used . This E.read-out . The E. promoter . The pPKSaccharomyces cerevisiae  and dried overnight at 37 °C. After washing with PBST (3 × 150 µL/well) the wells were blocked with BSA  and incubated at 37 °C for 2 h. Following this, the microtiter plates were washed with PBST (2 × 150 µL/well) and PBS (150 µL/well) [Black 96-well microtiter plates  were incubated with mannan from µL/well) .m in carbonate buffer, 120 µL/well) and incubated at ambient temperature and 100 rpm for 2 h. Following this, the microtiter plates were washed with PBST (2 × 150 µL/well) and PBS (150 µL/well) [For glycoarray preparation, black Immobilizer Amino™ F96 MicroWell™ plates (Nunc) were incubated with solutions of the respective glycosides in carbonate buffer  at ambient temperature and at 100 rpm overnight. Following washing with PBST (3 × 150 µL/well), the wells were blocked with ethanolamine (10 mµL/well) .a) Variation of bacteria concentration: Glycoarrays were prepared with 10 mm of the respective glycoside in carbonate buffer and to these glycoside-functionalized microtiter plates PBS buffer was added (100 µL/well). Then, the prepared bacterial solution (OD600 0.4) was added to well 1 and 2 and serially diluted (starting with well 2). The plate was incubated for 1 h at 37 °C, at 100 rpm, washed with PBS (3 × 150 µL/well), and then PBS was added (100 µL/well) and the fluorescence intensity (485 nm/535 nm) determined [(termined .b) Variation of glycoside concentration: Glycoarrays were prepared using serial dilutions of the respective glycoside (starting with 200 mm glycoside solution in carbonate buffer). Following this, the prepared bacterial solution  was added and the plate incubated for 1 h at 37 °C at 100 rpm. The plate was washed with PBS (3 × 150 µL/well), and then PBS was added (100 µL/well) and the fluorescence intensity (485 nm/535 nm) determined [(termined .m carbonate buffer except for pAPMan: 10 mм) and serial dilutions of the inhibitor solution added to the mannan-coated microtiter plate wells. The prepared bacterial solution  was added and the plate incubated for 1 h at 37 °C and 100 rpm. The plates were washed with PBS buffer (3 × 150 µL/well), and then the wells were filled with PBS (100 µL/well) and the fluorescence intensity (485 nm/535 nm) was determined [Solutions of the respective inhibitory glycosides were prepared (200 mtermined .pAPMan: 10 mм) and mixed with the prepared bacterial solution . /well) for preincubation. These mixtures were incubated for 1 h at 37 °C and 100 rpm and were then transferred to mannan-coated microtiter plates followed by incubation for 1 h at 37 °C and 100 rpm. The plates were washed with PBS buffer (3 × 150 µL/well), the wells filled with PBS (100 µL/well) and the fluorescence intensity (485 nm/535 nm) was determined [Serial dilutions of the respective inhibitory glycoside solution were prepared (starting concentration 200 mм in carbonate buffer except for termined .m in carbonate buffer) and mixed with a bacterial solution . The resulting mixtures were incubated for 1 h at 37 °C and 100 rpm. Following this, 50 µL of each mixture were transferred to an agar plate, which were incubated overnight at 37 °C. Bacterial growth was determined by visual inspection of the agar plates.Serial dilutions of the respective inhibitory glycoside solutions were prepared . On the other hand, a constant number of fluorescent E. coli cells were used in the adhesion experiments. Fluorescence read-out showed a plateau of maximal adhesion efficiency at “densely” functionalized plate surfaces and diminishing bacterial adhesion starting at lower glycoside concentrations when mannosides 1 or 2 were used for the functionalization of the microtiter plates (3 or 4) or galactosides (5 or 6), respectively, did not lead to an adhesive surface.Thus, glycoside solutions used for glycoarray fabrication were serially diluted starting with a comparatively high concentration , were now used as soluble inhibitors of type 1 fimbriae-mediated bacterial adhesion in solution. It was important to test, if the effect of the putative FimH ligands in solution was different from their effect as immobilized ligands. Hence, serial dilutions of inhibitor solutions were used to interfere with bacterial adhesion to mannan-coated microtiter plates. On each individual plate, we tested for methyl α-d-mannopyranoside (MeMan) to provide a universal reference inhibitor of type 1 fimbriae-mediated bacterial adhesion. Inhibition curves were determined for each tested inhibitor and IC50 values were deduced where possible. The IC50 value represents the inhibitor concentration at which 50% of bacterial adhesion is prevented. As MeMan was always simultaneously tested, relative inhibitory potencies  can be specified for all tested glycosides with respect to the IC50 obtained for MeMan, which was defined as reference inhibitor potency with IP≡1.In the next step of our study, all glycosides, which had been used before for glycoarray preparation  a soluble inhibitor has to compete against a highly mannosylated (mannan-coated) surface for binding to the type 1 fimbrial lectin. In contrast to this, in the preincubation-inhibition-adhesion assay [E. coli were first incubated with the respective inhibitor under physiological conditions for 1 h. Only then, this suspension was transferred to the mannan-coated microtiter plate surface. In the adhesion-inhibition assay both ligands, mannan and the tested inhibitor, are presented to the bacteria at the same time. In contrast, preincubation of the bacteria with inhibitor solution can lead to saturation of the lectin’s carbohydrate binding sites prior to the adhesion experiment on the mannan-coated surface. Thus, in this assay bacterial binding to mannan is only possible if the fimbrial binding site is not effectively saturated with the inhibitor after preincubation.In the adhesion-inhibition assay  are cytotoxic at high concentrations. However, all aminothiahexyl glycosides  were cytotoxic at high concentrations, the glucoside 4 at concentrations ≥100 mm, galactoside 6 at concentrations ≥50 mm, and, surprisingly, mannoside 2 inhibits bacterial growth even at a concentration of 25 mm.As a next step, it was important to analyze the effect of galactoside manno-, but also the gluco- and galacto-series (1–6) were investigated with type 1 fimbriae-mediated bacteria. Two different aglycone moieties were used, namely an aromatic p-aminophenyl unit and the 6-aminothiahexyl spacer (3 and 4) and galactosides (5 and 6) were tested in solution as inhibitors of bacterial adhesion to a mannan surface, rather unexpectedly, an inhibitory effect was observed  inhibit bacterial growth at high concentrations. Thus, the measured inhibitory effect could be shown to stem from toxicity rather than from carbohydrate-specific inhibition of adhesion. The question of why mannoside 2 shows the highest toxicity in this series cannot be answered at this stage, as E. coli bacteria have transporters for all three types of sugars, glucose, galactose, as well as mannose [To analyze the specificity of these effects, bacterial growth was tested after incubation with the employed glycosides at high concentrations . In this mannose . Recentl mannose . Interes mannose ,58,59,602. On the other hand, when glycosides such as 2, 4, or 6 are used to decorate surfaces, the resulting glycoarrays exert no cytotoxicity, suggesting that cellular uptake is a prerequisite for the effect (cf. Our study has important implications for further utilization of aminothiahexyl glycosides, regardless whether they are used as specific inhibitors or as control compounds in solution. As the aminothiahexyl aglycone is especially easily accessed in synthetic organic chemistry using the “thiol-ene” reaction of cysteamine to allyl glycosides, respective compounds are quite popular . It has fect cf. .E. coli with the highly mannosylated polysaccharide mannan. In contrast to this, in the preincubation assay the bacteria are initially exclusively presented to the glycoside ligand, and no competing compound is present in the first step of the assay. In accordance with these considerations, the IC50 of mannoside 1 is ~0.6 mm in the adhesion-inhibition assay, but 0.035 mm in the preincubation-inhibition-adhesion assay. An analogous observation was made with mannoside 2. In addition, the IC50 of the glucoside 3 is ~120 mm in the first assay and ~50 in the second.The effect of the assay set-up can be seen when data, which were obtained in the adhesion-inhibition assay are compared with those measured in the preincubation-inhibition-adhesion assay . In the At this end it remains unclear, whether conformational rearrangement of FimH can result in altered carbohydrate specificity. In the herein performed tests, a “relaxed” conformation of the FimH carbohydrate-binding site must be assumed. As a next step, we will perform adhesion studies under tensile force, based on the herein reported results. After allosteric activation of FimH according to the catch bond mechanism, carbohydrate specificity of FimH will be tested accordingly.


The isolation and purification of AFGPs is laborious, costly, and often results in mixtures, making characterization difficult. Recent structural investigations into the mechanism by which linear and cyclic AFGPs inhibit ice crystallization have led to significant progress toward the synthesis and assessment of several synthetic mimics of AFGPs. This review article will summarize synthetic AFGP mimics as well as current challenges in designing compounds capable of mimicking AFGPs. It will also cover our recent efforts in exploring whether peptoid mimics can serve as structural and functional mimics of native AFGPs.Antifreeze proteins (AFPs) and glycoproteins (AFGPs), collectively called AF(G)Ps, constitute a diverse class of proteins found in various Arctic and Antarctic fish, as well as in amphibians, plants, and insects. These compounds possess the ability to inhibit the formation of ice and are therefore essential to the survival of many marine teleost fishes that routinely encounter sub-zero temperatures. Owing to this property, AF(G)Ps have potential applications in many areas such as storage of cells or tissues at low temperature, ice slurries for refrigeration systems, and food storage. In contrast to AFGPs, which are composed of repeated tripeptide units (Ala-Ala-Thr) Choristoneura fumiferana [Tenebrio molitor [In order to survive in extremely cold environments, many organisms have evolved unique adaptive mechanisms. Nearly two-thirds of the surface of the earth consists of water, with the average surface temperature of seas and oceans varying from −2 °C to 30 °C depending on the latitude. Within the polar regions, seawater temperatures are consistently subzero because of the presence of dissolved salts. These subzero temperatures can be extremely harmful, if not deadly, to polar fish. As a cold-adaptation mechanism, various polar fish produce antifreeze proteins/peptides (AFPs) or antifreeze glycopeptides (AFGPs) to protect themselves from freezing damage. These proteins and peptides have ice-binding affinity, and can lower the freezing temperature of a solution non-colligatively without affecting its melting temperature, a phenomenon termed thermal hysteresis (TH). In addition, AF(G)Ps can inhibit ice recrystallization, in which large ice crystals grow at the expense of smaller ones, and thus prevent mechanical cell damage caused by ice regrowth. Currently, it is generally accepted that AF(G)Ps function through the adsorption of their flat ice-binding surfaces onto particular planes of ice crystals, and thus prevent or inhibit further ice growth ,2. AF(G)miferana  and Tene molitor  have a β molitor ,35.Pseudopleuronectes americanus) and shorthorn sculpin . To date, three-dimensional (3D) structures of the AFPs HPLC6 and ss3 have been solved [Among the several types of AFPs, Type I AFPs were the first reported and have been extensively studied. Type I AFPs are alanine-rich peptides found in blood samples from winter flounder  and its gene products possess Thr-O-linked glycosides. A typical AFGP consists of repeating tripeptide units, the alanyl-alanyl-threonyl (Ala-Ala-Thr)n=4–50 unit, connected to the disaccharide β-d-galactosyl-(1→3)-α-d-N-acetylgalactosamine through a glycosidic bond at the second hydroxyl group of the threonine residue. Eight distinct AFGP subtypes exist in nature. Among them, AFGP1 has the largest weight (33.7 kDa), and AFGP8 the lowest (2.6 kDa). In contrast to antifreeze glycoproteins, glycoproteins and glycopeptides are well known and extensively studied as biomolecules that play important roles in various biological processes such as immune differentiation, cell adhesion, cell differentiation, and cell growth regulation. Surprisingly, a tumor-associated carbohydrate antigen (T-antigen) found in mammals also contains the same glycosyl residue [The first AFGP gene was characterized from the Antarctic notothenioid ( residue .AF(G)Ps are believed to directly interact with the ice surface, resulting in freezing point depression to a level more than 200- to 300-fold greater than ordinary cryoprotectants (sugars and polyols) or salts, on a molar basis . The abii.e., thermal hysteresis (TH) assay [In the antifreeze activity assay of AF(G)Ps, two experimental techniques have been used, H) assay  and ice H) assay . In the H) assay . DSC proThe RI assay measures the size of ice crystals in AF(G)P solution using a microscope at frozen temperature. If AF(G)P has strong RI activity, smaller ice crystals are visible but control sample without AF(G)P shows large ice crystals during the process. This method is much simpler and faster than TH assay. However, it is more suitable to check the antifreeze activity is present or not because it is very difficult to obtain quantitative results.Pseudopleuronectes americanus) and shorthorn sculpin . The most extensively characterized type I AFP is the winter flounder HPLC6 AFP, which has 37 residues and high alanine content . It has three repeated sequences, which contain 11 amino acid repeats; Thr-Ala-Ala-X-Ala-X-X-Ala-Ala-X-X, where X can be any amino acid. The crystal structure of the HPLC6 AFP (PDB code 1WFA) [N- and C-terminal cap structures to ensure its stability in solution. The N- and C-terminal cap structures are stabilized by a hydrogen bond network that involves structurally ordered water molecules, and help the protein maintain its helicity (Type I AFPs have single α-helices and are found in winter flounder (de 1WFA)  revealedhelicity A. PrevioShorthorn sculpin ss3 AFP is another Type I AFP, which also has high alanine content  . Unlike Pseudopleuronectes americanus) with a molecular weight of 17 kDa. This protein is five times larger than HPLC6 AFP, and it exhibits significantly high TH activity even at low concentrations. Hence, this protein was named as hyperactive type I AFP [AFP9 from winter flounder is a longer type I AFP compared with HPLC6 AFP and has 4 repeat sequences . The TH pe I AFP . Althougpe I AFP .N and/or C-terminal residues were chemically synthesized, and their antifreeze activities examined. Furthermore, to investigate the relationship between the α-helicity and antifreeze activity of the peptides, the circular dichroism (CD) spectra of the peptides were analyzed. The decreased α-helical content of these peptides directly correlated with the decrease in their antifreeze activity. These results showed that the α-helical conformation is essential for the antifreeze activity of Type I AFP. To improve solid-phase synthesis and aqueous solubility, Haymet et al. have incorporated 2 salt bridges (A7K/A11E and A29K/A33E) into HPLC6 AFP. The mutants retained similar TH and recrystallization inhibition (RI) activities compared with wild-type HPLC6 AFP, but their solubilities were improved by the salt bridges [To identify key residues for ice binding, structure-activity relationship studies were performed on HPLC6 AFP using truncated or synthetic analogs. To date, over 50 mutant peptides of HPLC6 AFP have been produced and their antifreeze activities have been compared to that of wild-type HPLC6 AFP. It was initially suggested that Thr residues are critical for hydrogen bond interactions with water molecules on the ice surface. To test this hypothesis, Thr residues in HPLC6 AFP were replaced with Ser (T13S and T24S double mutant), which was expected to maintain the hydrogen bond with ice through its hydroxyl group. Unexpectedly, the mutation significantly reduced the TH activity. On the other hand, Thr to Val mutants (T13V and T24V double mutant) retained most of the TH activity (85% TH activity compared to that of wild-type HPLC6 AFP), indicating that the hydrophobic methyl group of Thr is important for ice binding in HPLC6 AFP . TherefoUnlike HPLC6 AFP, shorthorn sculpin AFPs contain no repeat spacing of Thr; however, shorthorn sculpin AFPs have several Lys and Arg residues . Initial modeling studies showed that the positively charged residues can be matched with the ice crystal lattice, indicating the participation of the charged residues in ice binding of shorthorn sculpin AFPs . For theThe ice-binding residues in hyperactive type I AFP have not yet been identified by mutation analysis, but sequence analysis and modeling studies have provided a possible ice-binding mechanism for hyperactive type I AFP . The strIn conclusion, the various type I AFPs have a common α-helical structure and many Ala residues, which constitute an Ala-rich hydrophobic ice-binding face. On the other hydrophilic side of the helix, charged or polar residues are involved in enhancing the solubility of Type I AFP.The unique physiochemical properties of AFGPs have led to considerable interest in these molecules for various applications; however, the lack of progress in their mechanistic and SAR studies is largely because of the presence of numerous isoforms of AFGP in blood plasma and the difficulty in isolating them in pure form . Moreovei.e., n = 10–12), but did not report any of their TH, ice growth habit modification, or recrystallization inhibition properties. These synthetic antifreeze glycoproteins (syAFGP) were prepared by directly polymerizing an unprotected glycopeptide macromonomer in the presence of diphenylphosphorylazide (DPPA) as a promoter  residue and a C-terminal thioester moiety, and a glycopeptide containing an N-terminal cysteine (Cys) residue, (2) conversion of the N-terminal Thz moiety to an N-terminal Cys residue, and (3) desulfurization of the Cys-containing glycopeptides to afford native and homogeneous AFGPs [Specifically, the effectiveness of this strategy relies on 3 key reactions: (1) native chemical ligation between a glycopeptide bearing an us AFGPs .d-galactosyl--α-d-N-acetylgalactosamine was substituted by β-d-galactosyl--α-d-galactose was reported in 1988 by Anderson and coworkers -3-O--l-threonine as a symmetrical anhydride [et al. [N-acetyl group, and with a substitution of the threonine residue in the alanyl-threonyl-alanyl (Ala-Thr-Ala) polypeptide with serine. Moreover, an interesting feature is the use of pentafluorophenyl ester (Pfp) to protect the C-terminus of the building block and benzoyl instead of acetyl groups to protect the hydroxyls. The Pfp ester is stable under glycosylation reaction conditions but also reactive enough to be utilized directly in solid-phase synthesis [Related solid phase synthesis procedures for AFGPs containing single sugars were reported by Filira d Jensen ,77 by emd Jensen ,77. The  [et al.  was the nhydride . SimilarC- and N-terminus on TH activity -α-d-N-acetylgalactosamine in ice recrystallization activity by studying closely related structures, including those of β-linked -, -, and -galactosyl-N-acetyl galactosamine and the β--galactosyl-galactoside  close to the sugar residue, as well as the length of the side chain bearing the triazole linkage, plays key roles in the activity [Another flexible platform reported by three different research groups for the synthesis of a wide range of antifreeze glycopeptide analogues used click chemistry as an alternative. In the first convergent synthesis reported by Miller et al. , triazol unknown . In the n [et al . The anan [et al  -containing glycopeptoid derivatives. Peptoids can be described as mimics of α-peptides in that the side chain is attached to the backbone amide nitrogen instead of the α-carbon. Because of their ability to confer high conformational flexibility, high structural diversity, and improved proteolytic stability, peptoids have been developed as a new peptide modification strategy. In order to generate metabolically stable glycopeptide analogues without impairing antifreeze activity, we reported a highly efficient convergent strategy in which the glycosylated peptoid building blocks  or Nα-Fmoc-Ala-NSer ) were prepared using glycosylated Fmoc-protected peptoid monomer and the commercially available Fmoc-l-Ala-OH in the presence of DIC. The key glycosylation reaction was carried out using an Fmoc-protected peptoid monomer (Fmoc-NThr-OtBu) with 3,4,6-tri-O-acetyl-2-azido-α-d-galctopyranosyl bromide in the presence of Ag2CO3 [N-linked glyco-threonine were synthesized and tested for RI activity. In accordance with previous findings, native cyAFGP showed strong RI activity even though the disaccharide was replaced with a monosaccharide, whereas the peptoid derivatives (cypAFGPs) did not show any activity [n), ether (Ac(Nme)n), and methyl (Ac(Sar)n) substituents were synthesized to compare the effects of backbone structure, side chain chemical composition, and sequence length on antifreeze activity Ps are considered less toxic or nontoxic cryoprotective agent (CPA) candidates because they depress the freezing point non-colligatively, so that a much lower amount could be used than in the case of toxic chemical CPAs. In this section, the state of research into the development of AFP-based preservation or cryopreservation studies will be described.AF(G)Ps can decrease the freezing temperature of solutions and inhibit ice recrystallization during freezing and thawing procedures . These uIn general, the cryopreservation of cell lines using general cryoprotectants is well established. The high amount of CPAs used in the cryopreservation of cells and tissues diminishes freezing damage, but also results in cytotoxicity . Even thin vivo-matured metaphase II oocytes [Crassostrea gigas) oocytes [E. coli [AF(G)Ps could play a role in the survival of organisms by preventing damage caused by recrystallization. Many attempts to develop promising hypothermic storage or cryopreservation techniques have been carried out on bovine and porcine oocytes ,102, mou oocytes , vertebr oocytes ,107, ani oocytes , oyster  oocytes , carp sp oocytes , ovine s oocytes , sheep e oocytes , bull sp oocytes , seabrea oocytes , chimpan oocytes , zebrafi oocytes , rice em oocytes , human e oocytes , islet c oocytes , platele oocytes , E. coli[E. coli , and pig[E. coli .It has also been proposed that the cryopreservation property of AF(G)Ps may come from the interactions of proteins with the cell membrane ,123,124,RBCs have a significant impact on both the military and civilian communities with respect to transfusion. Because RBCs were first stored using citrate and glucose in 1916 , other sOrgans can be stored hypothermically up to 72 h depending on the organ type, but they undergo gradual deterioration during cold storage ,148. SevEven though the problem of this negative effect of AFGP has not been solved, this issue could possibly be addressed by peptide or protein engineering through modifying the needle-like ice structuring property of AFGP. This technology might not be readily available before fully understanding the structure-function relationship of AFGP, but it is promising as results of synthetic analogues are being accumulated.Considering the fact that AFP-induced needle-like ice crystals destroyed the cells , AFPs haThe ice RI activity of AFP has been successfully applied to food preservation. Studies have reported that AFPs are effective in preserve food texture through ice recrystallization inhibition during freezing and storage ,155,156.The AFP gene may confer the property of cold tolerance to transgenic organisms. Previous studies have shown that transgenic salmon constructed by the introduction of type I AFP into eggs revealed freeze-resistant traits, and tissue-specific seasonal AFP expression was identified ,161. A tC-linked Xaa-Gly-Gly mimics have delivered novel compounds that display interesting RI properties and low toxicities towards human embryonic liver and kidney cells but do not suppress the freezing temperature or inhibit nucleation in the same way that biological antifreeze compounds do. In contrast to the activity of triazole- and N-linked peptoid mimics, these compounds are also stable against hydrolysis and amenable to the scale up production of limited number of amino acid coupling using solid phase synthesis. Further structural modifications on these mimics in terms of optimizing flexibility could modulate their antifreeze properties. A recent study on the stabilization of membranes during the cooling of phospholipid membranes using AF(G)Ps indicates that higher molecular weight AFGPs confer increased protection to the cell membranes [Despite over 30 years of studying antifreeze (glyco) proteins, there are still several issues such as cytotoxicity, low abundance in nature, and stability that must be overcome before AFGPs can find widespread application. First, a significant limitation of native AFGP is dynamic ice shaping/TH , which exacerbates cellular damage by altering crystals to form long specular ice crystals ,170,171.embranes . In this