Planktothrix-like сyanobacteria has been often reported for shallow eutrophic\hypertrophic lakes in central Europe in summer\autumn. However studies on phytoplankton growth under ice cover in nutrient-rich lakes are very scarce. Lake Nero provides a good example of the contrasting seasonal extremes in environmental conditions. Moreover, the ecosystem underwent a catastrophic transition from eutrophic to hypertrophic 2003–05, with dominance of filamentous cyanobacteria in summer\autumn. Towards the end of the period of ice cover, there is an almost complete lack of light and oxygen but abundance in nutrients, especially ammonium nitrogen, soluble reactive phosphorus and total phosphorus in lake Nero. The aim of the present study was to describe species composition and abundance of the phytoplankton, in relation to the abiotic properties of the habitat to the end of winters 1999–2010. We were interested if Planktothrix-like сyanobacteria kept their dominant role under the ice conditions or only survived, and how did the under-ice phytoplankton community differ from year to year.The permanent dominance of Limnothrix, Pseudanabaena, Planktothrix) were present in all winter samples but did not always dominate. Favourable conditions included low winter temperature, thicker ice, almost complete lack of oxygen and high ammonium concentration. Flagellates belonging to Euglenophyta and Cryptophyta dominated in warmer winters, when phosphorus concentrations increased.Samples collected contained 172 algal taxa of sub-generic rank. Abundance of phytoplankton varied widely from very low to the bloom level. Cyanobacteria (Planktothrix - like сyanobacteria in summer/autumn may follow several scenarios in the end of winter. It may be intense proliferation сyanobacteria normally dominating in summer, or the switch to the other species like the euglenoids and cryptomonads flagellates, or almost total depletion of phytoplankton.A full picture of algal succession in the lake may be obtained only if systematic winter observations are taken into account. Nearly anoxic conditions, severe light deficiency and high concentration of biogenic elements present a highly selective environment for phytoplankton. Hypertrophic water bodies of moderate zone covered by ice in winter and dominated by  In temperate lakes, conditions for the development of phytoplankton in winter differ greatly from those in summer. Conventional models of phytoplankton seasonal succession assume a minimum of phytoplankton biomass under ice, due to a combination of unfavorable factors, especially low temperature, short days and poor light penetration - unless the ice is almost transparent. These winter survivors give rise to a new algal community providing the earlier start advantage to the aboriginal species and thereby influencing summer species composition. The degree of under-ice depletion of algal community remains understudied, although some studies of shallow eutrophic lakes in the more temperate climatic zones of Europe and America, emphasized the importance of contrasting conditions under the ice and of the open-water periods for annual algal succession -10.Nevertheless, a few early studies noted mass development of phytoplankton under clear ice. Thus, Yasnitsky’s  study inPlanktotrichetum - S1 codon. Species that make up this assemblage  Meffert, Pseudanabaena limnetica (Lemm.) Kom.), share the trait of dispersion in the plankton as solitary filaments, have superior light interception properties, and a propensity to extreme photoadaptation in turbid, oligophotic conditions. These species maintain a near steady-state within the phytoplankton community of eutrophic/hypertrophic lakes [Planktotrichetum codon dominating in summer.This points at the possibility of special importance of the under-ice period in maintaining of a lake’s ecological situation as a whole. Lake Nero  provides a good example of the contrasting seasonal extremes, allowing a detailed study of the link between summer algal communities and the under-ice biota. The lake has been intensely studied during the past few decades -22. Lakeic lakes . A suddeic lakes ,22. HereLake Nero is covered by ice from November-December to April-May. Thus, the period of ice cover may last for 148–211 days. Annual mean temperature in the Yaroslavl Province grew approximately by 1.2°C during last 50 years. Simultaneously winter temperature increased by 1.7°C. Precipitation increased too. All these changes caused steady water level increase of the Lake Nero observed since 1976. The lake's surface clears from ice now by five days earlier than half a century ago. The dates of freezing did not change . There i2 remain and the water smells of sulphide. In approximately half of all the samples collected from under the ice, oxygen was not detected at all. In 2003 and 2005, mass fishkill occurred. In the milder winters, openings in the ice cover often appeared. Detectable amounts of methane and CO2 are vented through these openings. They may also increase slightly the input of oxygen into the water. In 2008, a large (ca 20 m wide), stable opening developed as a consequence of industrial activity on the shore. Yet, in samples collected only 10 m from the opening, oxygen concentration remained low (1.6 mg L-1).Oxygen is rapidly consumed under the ice so that, before the ice finally melts in spring, only trace concentrations of OThe under-ice concentrations of other biogenic elements are generally at their highest annual levels at this time ,25. Non-Over the full period of our studies, 172 algal taxa of sub-generic rank were encountered in samples collected from under ice in Lake Nero  Kütz., Aphanizomenon gracile (Lemm.) Lemm., Scenedesmus communis Hegewald, fall to low levels in the winter. The only species that was found in all winter samples was Limnothrix redekei. Pseudanabaena limnetica was present in about 80% of samples. The frequencies of Planktothrix agardhii, Euglena acus Ehr., Cryptomonas ovata Ehr. and Aulacoseira ambigua (Grun.) Sim. were each about 70%. Other species were found in less than half of the samples. The species found only in winter samples were: the cyanophyte Pseudanabaena acicularis (Nyg.), the dinophyte Woloszynskia tenuissima (Lauterb.) Thomp, the chlorophyte Chlorogonium maximum Skuja, the cryptophyte Сhroomonas minima Czosnow and the euglenophyte E. acus var. minor Hansg.The composition of the phytoplankton community under ice generally involved species that are present as vegetative stocks through most of the year. β and β-mesosaprobic forms.From eco-geographical point of view the under-ice phytoplankton of Lake Nero is a cosmopolitan one with some prevalence of planktonic species indifferent to salinity; there is a significant presence of oligo-The Sorensen coefficient of compositional similarity between winter algal communities in different years varied from 9.2 to 33.3, indicating significant changes of species compositions in winter.-1 or 3 mg L-1 . In most cases the biomass was higher near to the bottom. Average cell volume varied according to the species composition. There were no significant dependencies between total phytoplankton biomass under the ice and in May as well in September. The same holds if only S1 codon biomass is taken into account.The total abundance of phytoplankton under the ice and the Shannon index of diversity, as calculated from the biomass values, varied through the winter Table. . Most fr Figures  and 3. Ta under ice varied together with the biomass  was found in March 2008 at the sampling point N 8. Much more commonly, values fell between 1 and 3 μg L-1, coinciding with low abundance of phytoplankton in winter. The high concentration of the phaeopigments was a typical feature of phytoplankton collected under the ice, accounting for 40 – 75% of the measured total Chl а. This probably reflects a high proportion of moribund or dead cells.The concentrations of chlorophyll s Figure A. The ma-1. As a rule Limnothrix redekei was dominant in the samples of high cyanophyta biomass. Pseudanabaena limnetica, Pseudanabaena acicularis, Planktolyngbya limnetica were less often abundant in these samples. Usually, the Planktotrichetum organisms were evenly distributed across the water column  were found to be the main components of the under-ice community. Diatoms, Dinophyta or green algae dominated or subdominated the communities only rarely Figure B. The len Figure .-1, with the exception of March 2008 when their absolute biomass was between 3.6 - 21.3 mg L-1 with Euglena limnophila var. swirenkoi domination. The vertical distribution of euglenoids varied from year to year: for example, in 1999, the maximum recorded proportion of near-bottom biomass was 91.6%; in 2004, their abundance in the upper layer was 5 times higher than near the bottom.Euglenophyta were less abundant than Cyanophyta. Its biomass varied at 0.002 - 0.5 mg L-1. In March 2008, together with the Euglenophyta, the proliferation of this group was exceptionally high under the ice: from 8.6 to 13.4 mg L-1, with dominant Cryptomonas rostrata Troitzkaja emend. I. Kis. Their vertical distribution peaked in the near-bottom area  Lemm., Chlamydomonas sp. and Pteromonas variabilis Hub. – Pest. Green algae mostly occurred close to the surface  revealed clearly defined groups of samples  of biotic parameters  and abiotic parameters , althoural Asia , the Rybral Asia ,36 and sral Asia .Planktothrix - like cyanobacteria under the ice may depend on the adaptive abilities to very low light incomes and the tolerances to low temperatures [Planktothrix agardhii through all winter. In Lake Wolderwijd (Holland), Planktothrix agardhii, Limnothrix redekei and Pseudanabaena limnetica are vegetative almost all year round [Planktothrix - like cyanobacteria is typical for Lake Nero, even during the coldest winters, that could be supported the hypothesis of C. Wiedner and B. Nixdorf. Coexistence of low amounts of the S1 codon species through all winters including very low levels biomass confirms the importance of the functional group concept in phytoplankton ecology.The abandance of the eratures . In Lakeeratures  detectedar round . Some auar round ,39. Wiedar round  investigar round . The preHigh proliferation of flagellates belonging to the Euglenophyta and Cryptophyta and the high frequencies in samples, may be regarded as a peculiarity of the under-ice period for Lake Nero too and allows one to treat them from the point of view of functional groups . The prePlanktorhrix – like cyanobacteria or of the species of W2-Y flagellates. The statistical analysis suggests that, during the coldest winters under thick ice and anoxic conditions, and in the presence of high ammonium nitrogen, S1 enjoy a considerable functional and selective advantage. Conversely, warmer winters and higher phosphorus concentrations are more favorable to the W2-Y euglenoids and cryptomonads flagellates.In Lake Nero, the fraction of flagellates (W2-Y codon) was a small part of the biomass in the years when S1 algae were blooming. This observation is supported statistically. Therefore, the composition of the winter phytoplankton in Lake Nero, involves either the proliferation of species of S1 Planktothrix-like cyanobacteria (S1 codon) which is observed from early summer to late autumn may persist through winter and even a small amount of winter survivors belonging to the summer dominants are sufficient for their reestablishment in summer. However here we show that the switch of the dominant organism to the euglenoids and cryptomonads (W2-Y codons) is likely to occur.This study provides evidence that conditions of near anoxia, of severe light deficiency and high concentration of biogenic elements in the end of winter in the hypertrophic lake present highly selective conditions for proliferation of most phytoplankton. The abundance of the phytoplankton varies from trace amounts to an under-ice bloom level. A full picture of algal succession in the lake may be obtained only if systematic winter observations are taken into account. The dominant role of 2; its maximum depth is 4.7 m; mean depth is 1.6 m. The lake was formed in the Late Pleistocene period, ca. 150 000 years ago, during the wasting of the Moscow Glacier. During the last 5000 years, starting at the end of the Boreal period or the beginning of the Atlantic Period, the lake has been shallow, inhabited by species typical of eutrophic water bodies. Lake Nero has 12 inflowing rivers, the largest of which is Sara River. There is one outlet to the lake: Veksa Rostovskaya River, which flows to the Kotorosl’ River and which is, in turn, a tributary of the Volga River. The water level has been regulated by the dam from the end of 1980. The area of the watershed is 1314 km2; the water exchange rate is 1,9 times per year. The chemical composition of the lake differs from that of other lakes of the area: it contains more minerals , with a high concentration of chloride [Lake Nero is situated in the Upper Volga River Basin. It is the largest lake in Yaroslavl province; its area is about 58 kmchloride . The botchloride .a) and biogenic elements were done from one bathometer. Samples for chemical analysis were fixed in the field with chloroform and processed on return to the laboratory. Soluble phosphorus was determined colorimetrically using the sulfate / molybdate method; ammonium, nitrate and nitrite nitrogen were determined using Nessler reagent, sulfaphenol and Griss chemical [a and phaeopigments were calculated according to the method and equations of Lorenzen [The samples were collected under-ice in March of 1999, 2002–2010 from one to three stations, situated in Northern and Central parts of the lake, close to the city of Rostov Figure . Contraschemical . PhotosyLorenzen . PhytoplLorenzen -49. ShanLorenzen  was useda+b is the number of species in common detected in the samples a and b, and Na and Nb is the number of species in the samples a and b.where NThis study is based on analysis of 32 samples and sets of limnological observations. Coefficients of correlation and factor analyses were solved using conventional methods as adopted in the STATISTICA software, version 6.0  and MS Excel programs.The author’s declare that they have no competing interests.a) and of elements of biolological importance and co-wrote the manuscript. SSch was involved in the flora characterization as well in the description of the habitat and co-wrote the manuscript. All authors have read and approved the final manuscript.BO did the field work, the identification and counting of phytoplankton, performed the statistical analysis and wrote the manuscript. SS made the analysis of pigments .Click here for file


We report the case of a 17- year-old male that suffered from a systemic anaphylactic episode during exercise fifty minutes after the ingestion of mushrooms cooked with wine in a restaurant. This anaphylaxis occurred at the age of 16 when the patient was playing handball indoors with a high level of humidity. The patient stopped mushrooms ingestion. There were no symptoms when the exercise occurred after the ingestion of other kinds of food, and no more anaphylactic episodes occurred. This patient suffered from persistent rhinosinusitis since the age of 10 years old.Agaricus bisporis), total IgE and specific IgE determinations (sIgE) to airborne allergens were also performed, according to case history. The molecular mass of the IgE binding bands was calculated by means of SDS PAGE immunoblotting. In order to study the presence of cross reacting IgE in the patient serum we carried out a SDS-PAGE immunoblotting-inhibition assay using mushroom extract in solid phase and Alternaria alternata as inhibitor. An oral challenge test (OCT) with the restaurant mushroom dish was performed.Skin prick tests (SPT) to commercial extracts of airborne allergens and food allergens were carried out. Prick-to-prick tests (PP) to the mushroom eaten by the patient before the reaction , Cladosporium herbarum (4.43 kU/L), Curvularia lunata (3.41 kU/L), Aspergillus fumigatus (2.74 kU/L), and Candida albicans (1.71 kU/L).SPT was positive to pollen from grasses, cypress, alder and cedar as well as to Alternaria alternata was used as inhibitor. The OCT with mushroom was negative and the patient refused an OCT with exercise after the ingestion of mushroom.Immunoblotting assays revealed two IgE binding bands of nearly 30 kDa in the mushroom extract. A total inhibition of these two IgE binding bands was detected when Alternaria alternata was confirmed. There are a few references in the literature concerning cross-reaction between mushroom and moulds in patients with fungal allergy, particularly with rhinosinusitis. Since the OCT with mushrooms cooked in the same way as the day of the allergic reaction, was negative several factors might play a role in this reaction such as exercise performed indoors in a high level of humidity environment, with moulds.Immunologic cross-reactivity between mushroom and I Carrapatoso: None declared, B Bartolome: Employee of 2 R&D Department, Bial-Aristegui, Bilbao, Spain, E Faria: None declared, F Ribeiro: None declared, A Segorbe Luís: None declared.


NF-E2 related factor-2 (Nrf2) promotes the transcription of many cytoprotective genes and is a major drug target for prevention of cancer and other diseases. Indeed, the cancer-preventive activities of several well-known chemical agents were shown to depend on Nrf2 activation. It is well known that chemopreventive Nrf2 activators stabilize Nrf2 by blocking its ubiquitination, but previous studies have indicated that this process occurs exclusively in the cytoplasm. Kelch-like ECH-associated protein 1 (Keap1) binds to Nrf2 and orchestrates Nrf2 ubiquitination, and it has been a widely-held view that inhibition of Nrf2 ubiquitination by chemopreventive agents results from the dissociation of Nrf2 from its repressor Keap1. Here, we show that while the activation of Nrf2 by prototypical chemical activators, including 5,6-dihydrocyclopenta-1,2-dithiole-3-thione (CPDT) and sulforaphane (SF), results solely from inhibition of its ubiquitination, such inhibition occurs predominantly in the nucleus. Moreover, the Nrf2 activators promote Nrf2 association with Keap1, rather than disassociation, which appears to result from inhibition of Nrf2 phosphorylation at Ser40. Available evidence suggests the Nrf2 activators may block Nrf2 ubiquitination by altering Keap1 conformation via reaction with the thiols of specific Keap1 cysteines. We further show that while the inhibitory effects of CPDT and SF on Nrf2 ubiquitination depend entirely on Keap1, Nrf2 is also degraded by a Keap1-independent mechanism. These findings provide significant new insight about Nrf2 activation and suggest that exogenous chemical activators of Nrf2 enter the nucleus to exert most of their inhibitory impact on Nrf2 ubiquitination and degradation. Nrf2 is activated itself by many chemopreventive agents and is essential for some of these agents to prevent cancer and other diseases in animal models Nrf2 has emerged as a ubiquitous transcription factor that plays a critical role in the maintenance of cellular homeostasis. It stimulates the transcription of genes involved in many aspects of cytoprotection, most notably the Phase 2 genes, e.g., glutamate cysteine lygase (GCS) and NAD(P)H:quinone oxidoreductase-1 (NQO1). Indeed, Nrf2 knockout mice showed significantly increased susceptibility to a variety of diseases, such as cancer nrf2 gene transcription may be stimulated However, the mechanism by which chemopreventive agents activate Nrf2 remains less understood. While most studies have indicated that chemopreventive agents activate Nrf2 by blocking its degradation at the protein level, there are also studies suggesting that nrf2 gene transcription and Keap1 protein expression. We further show that CPDT and SF inhibited Keap1-dependent Nrf2 ubiquitination, but Nrf2 was also degraded via a Keap1-independent pathway. Neither CPDT nor SF disassociated the Nrf2-Keap1 complex or disrupted the ubiquitin ligase complex, but both compounds inhibited Nrf2 phosphorylation, which may account for the sustained association of Nrf2 with Keap1. Moreover, we demonstrate that the machinery for Nrf2 ubiquitination and proteasomal degradation exists in both cytoplasm and nucleus but that inhibition of Nrf2 ubiquitination by CPDT and SF occurs primarily in the nucleus, rather than in the cytoplasm. These findings significantly advance our understanding about the mechanism of Nrf2 activation and also have important implication for the development of Nrf2-based chemopreventive strategies. Our data indicate for the first time that Nrf2 chemopreventive agents enter the nucleus to inhibit nuclear Nrf2 ubiquitination.The present study was undertaken to further understand the mechanism of chemical activation of Nrf2. The study was carried out in multiple human and animal cell lines, utilizing CPDT and SF as prototypical Nrf2 activators. Both CPDT and SF  and NBT-II cells (a rat bladder cancer cell line) , indicatFigure S1).As expected, Nrf2 elevation by CPDT and SF was accompanied by significantly increased expression of GCS (heavy subunit) and NOQ1 (+/+ cells or RT-4 cells (pretreated with control siRNA) with either CPDT (50 µM) or SF (8 µM) for 6 h led to significant elevation of Nrf2 protein. In contrast, the inductive effects of CPDT and SF were either absent in MEF/Keap1−/− or greatly attenuated in RT-4 cells with Nrf2 knockdown  or Keap1 knockdown by siRNA in RT-4 cells resulted in increased expression of Nrf2 . Moreover, both CPDT and SF prevented wild type Keap1 and the C257S mutant from reducing Nrf2, but their effects on Nrf2 in cells transfected with either the C273S mutant or the C288S mutant were marginal. Given that both C273 and C288 were previously shown to be directly bound by dexamethasone 21-mesylate, a Nrf2 activator, through chemical reaction with the thiol groups Previous studies have identified two cysteine residues of Keap1 (C273 and C288) to be critical for Nrf2 stabilization by chemical activators Contrary to the wide-spread belief that inhibition of Nrf2 degradation by Nrf2 activators occurs exclusively in the cytoplasm, our experiments showed that inhibition of Nrf2 degradation by both CPDT and SF took place predominantly in the nucleus, as described below. Cytoplasmic and nuclear fractions were carefully prepared from both RT-4 cells and NBT-II cells after treatment with CPDT (50 µM) or SF (8 µM) for 6 h. No cross-contamination was detected, as the nuclear marker lamin A was not detected in the cytoplasmic fraction and the cytoplasmic marker GAPDH was not detected in the nuclear fraction  with anti-Keap1 or anti-Nrf2, we again found that neither CPDT nor SF had any effect on Keap1 protein level either in the cytoplasm or in the nucleus in both cell lines, but both agents significantly increased the Keap1-Nrf2 complex, mainly in the nucleus  for 24 h, followed by treatment with CPDT or SF for 6 h in the presence of MG132 (to inhibit degradation of ubiquitinated Nrf2), from which both cytosolic and nuclear fractions were prepared. We focused on NBT-II cells, because ectopic expression of ubiquitin (Ub) in RT-4 cells was extremely poor. IB analysis showed that sample preparation was satisfactory, as the cytoplasmic protein GAPDH was not detected in the nuclear samples and the nuclear protein lamin A was not detected in the cytosolic samples Figure 4We next asked how CPDT and SF prevented Keap1 from promoting Nrf2 ubiquitination. It has been previously shown that Keap1 and Nrf2 form a ubiquitin ligase core complex with Cul3 and Rbx1 in the cytoplasm ,,nrf2 gene transcription and Keap1 protein expression. Thus, the activities of these agents also fit with the widely accepted view that chemopreventive agents generally activate Nrf2 by blocking its protein degradation. Based on these considerations, CPDT and SF may be viewed as prototypical Nrf2 activators. It should be noted, however, that in all of our experiments, the Nrf2 band was detected at ∼100 kD, although the expected molecular weight of Nrf2 is ∼68 kD. The exact reason is not known, but this phenomenon has been previously reported and may be related to a Nrf2-actin complex In the present study, CPDT and SF were used as probes to understand the mechanism of chemical activation of Nrf2. CPDT and SF belong to two well-known classes of Nrf2 activators - dithiolethione and isothiocyanate, many of which are being investigated for prevention of cancer and other diseases. The chemopreventive activity of SF, which occurs in broccoli and other plants is particularly promising Keap1 promotes proteasomal degradation of Nrf2 by orchestrating Nrf2 ubiquitination. As expected, we found that Keap1 knockout or knockdown caused significant increase in Nrf2 level, whereas forced expression of Keap1 reduced Nrf2 expression Figure 2Figure S2) and ectopically expressed Ub existed largely in the cytoplasm in NBT-II cells , including anti-Nrf2 , anti-GCS , anti-Keap1 (sc-15246), anti-lamin A (sc-20680), anti-ubiquitin (sc-8017) and anti-His-tag (His-probe H3), and from Millipore , including anti-Rbx1 (AB3737) and anti-GAPDH (MAB374). Other antibodies including anti-NQO1 (3187), anti-p-Nrf2  and anti-Cul3 (611848) were purchased from Cell Signaling , Epitomics , BD Biosciences , respectively. An anti-His-HRP-conjugated antibody was purchased from Qiagen . CPDT was provided by Dr. Rex Munday −/−, MEF/Keap1−/−/Nrf2−/−) 2. All media were supplemented with 10% FBS. For treatment with the test agents, cells were seeded in 10-cm dishes (1–2×106 cells/10 ml medium/dish) or 6-well plates (0.25×106 cells/2 ml medium/well) overnight and then treated with a test agent for a desired time before harvest by trypsin treatment and low-speed centrifugation at 4°C. All agents were dissolved in DMSO, and the final DMSO concentration in the media was <0.25% (v/v). To knock down Keap1, cells were transfected with Keap1 stealth RNAi (HSS190639) or a universal control stealth RNAi (12935300) from Invitrogen  with lipofectamine 2000 for 48 h, following the manufacturer's protocol. For gene overexpression, cells were transfected with a desired plasmid (2 µg DNA/well for MEF or 6 µg DNA/dish for NBT-II cells) for 24–48 h, according to the manufacturer's recommended protocol for plasmid transfection with lipofectamine 2000.NBT-II cells Cytoplasmic and nuclear fractions were prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents Kit , following the manufacturer's instruction. For immunoprecipitation (IP), cytosols and nuclear extracts were incubated with a desired IP antibody, and the immune complexes were pulled down by incubation with protein A or G sepharose 4 Fast Flow Beads  and centrifugation. When measuring ubiquitinated Nrf2, the cytosols and nuclear extracts were denatured first to disrupt potential association of Nrf2 with other proteins before IP. These and other samples were electrophoresed on SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were blocked in TBS-0.05% Tween 20 with 5% non-fat milk, incubated with primary antibodies, and after extensive wash, incubated with a peroxidase-conjugated secondary antibody. Protein bands were visualized using either the Amersham ECL Plus System  or the SuperSignal West Pico Chemiluminescence Detection System (Thermo Scientific).5′-agt cgc ttg ccc tgg ata ttc-3′ and 5′- gcc gga gtc aga gtc att gaa-3′; human Nrf2, 5′-atg gat ttg att gac ata ctt t-3′ and 5′- act gag cct gat tag tag caa t-3′; GAPDH, 5′- gac cac agt cca tgc cat ca -3′ and 5′ – tcc acc acc ctg ttg ctg ta- 3′. PCR was carried out in a 50 µl volume and run for 30 cycles in an Eppendorf Mastercycler Gradient Thermal Cycler . The amplified products were electrophoresed on agarose gel and stained with ethidium bromide.Total RNA was isolated using the Qiagen RNeasy Mini Kit and Qiagen Qiashredder , from which cDNA was synthesized using random primers and SuperScript II RT (Invitrogen). PCR primers were as follows: Rat Nrf2, Figure S1Stimulation of Nrf2 transactivation actvitity by CPDT and SF. HepG2 cells were stably transfected with either a Nrf2 reporter construct (ARE-TK-GFP), where the cDNA coding the green fluorescence protein (GFP) was cloned in tandem behind the Nrf2-binding element antioxidant response element (ARE) and the thymidine kinase promoter (TK), or a control vector (TK-GFP) as previously described (TIF)Click here for additional data file.Figure S2The effects of CPDT and SF on Keap1-mediated Nrf2 degradation. Murine embryonic fibroblasts (MEF) were cultured in Iscove's modified DMEM. MEF with knockout of both Keap1 and Nrf2 were co-transfected with expression vectors of Nrf2 and one of the two Keap1 mutants (C257S and C273S) for 48 h, followed by treatment with vehicle, CPDT (50 µM) or SF (8 µM) for 6 h. Both cytosolic fractions and nuclear fractions were prepared, using the NE-PER Nuclear and Cytoplasmic Extraction Reagents Kit . Cross-contamination was ruled out by IB of β-tubulin (cytoplasmic marker) and lamin A (nuclear marker). Both fractions were then subjected to IP by anti-Keap1, followed by IB with anti-Nrf2 and anti-Keap1.(TIF)Click here for additional data file.


These results, considered in the known context of Cdks activity in neuroblasts, suggest that Cdk5 is the enzyme responsible for this phosphorylation. We find that the phosphorylation by PKC at the effector domain does not occur in the same molecules that are phosphorylated at serine 25. The in situ analysis of the subcellular distribution of these two phosphorylated MARCKS variants revealed that they are also segregated in different protein clusters. In addition, we find that a sustained stimulation of PKC by phorbol-12-myristate-13-acetate (PMA) provokes the progressive disappearance of phosphorylation at serine 25. Cells treated with PMA, but in the presence of several Ser/Thr phosphatase  inhibitors indicated that this dephosphorylation is achieved via a phosphatase 2A (PP2A) form. These results provide new evidence regarding the existence of a novel consequence of PKC stimulation upon the phosphorylated state of MARCKS in neural cells, and propose a link between PKC and PP2A activity on MARCKS.MARCKS (Myristoylated Alanine-Rich C Kinase Substrate) is a peripheral membrane protein, especially abundant in the nervous system, and functionally related to actin organization and Ca-calmodulin regulation depending on its phosphorylation by PKC. However, MARCKS is susceptible to be phosphorylated by several different kinases and the possible interactions between these phosphorylations have not been fully studied in intact cells. In differentiating neuroblasts, as well as some neurons, there is at least one cell-type specific phosphorylation site: serine 25 (S25) in the chick. We demonstrate here that S25 is included in a highly conserved protein sequence which is a Cdk phosphorylatable region, located far away from the PKC phosphorylation domain. S25 phosphorylation was inhibited by olomoucine and roscovitine in neuroblasts undergoing various states of cell differentiation  MARCKS (Myristoylated Alanine-Rich C Kinase Substrate) is a ubiquitous natively unfolded protein originally discovered as a major protein kinase C (PKC) substrate in the nervous system in vitro for proline-directed kinases, although the phosphorylated residues were not identified in vitro at serine 26 Sometime after the discovery of MARCKS and the ED phosphorylation, other six serine/threonine phosphorylated residues were found in MARCKS purified from membrane fractions of bovine brain, although its cell type origin was not determined in situ localization. The finding of some sequence singularities in the protein region where S25 lies, as well as the results from pharmacological assays, indicate that a Cdk is the kinase involved in the phosphorylation of S25. Using phorbol 12-myristate13-acetate (PMA) as a PKC stimulator we were also able to establish how, in these conditions, there is a disappearance of the phosphorylation at S25 and to localize differently phosphorylated MARCKS protein clusters in isolated cells. We also found that MARCK S25 dephosphorylation depends on the activity of a phosphatase inhibitable by calyculin A, but not by tautomycetin or other classical protein phosphatase inhibitors.On one hand, MARCKS has been studied in many different cell types and has been implicated in cross talks between some signal transduction pathways. On the other hand, there is a substantial amount of information concerning its role in secretory and migration activities involving components of the cell cortex  for variable times, as indicated in each figure. For control experiments, cells were incubated with vehicle (DMSO) or NaCl (control for LiCl). For PKC stimulation experiments, E8 neural retina cell suspensions were cultured three days in DMEM with 10% FCS, and then treated with PMA  or DMSO at the concentrations, and for the times, indicated in each figure. For phosphatase inhibitor treatments, cells were first washed in DMEM without FCS and then incubated in this medium containing okadaic acid , cyclosporin A , calyculin A , FK506 , or tautomycetin  at different concentrations and for variable times, as indicated in each figure and in the text. For control experiments, cells were incubated with vehicle (DMSO) at the appropriate concentration. For PKC stimulation, PMA was added to the cell cultures without removing the media containing the phosphatase inhibitors or vehicle. After incubation, isolated cells were lysed in electrophoresis buffer, containing SDS and β-mercaptoethanol. For microscopic examination, cells were plated on glass coverslips pre-coated with poly-L-ornithine or laminin . After incubation they were fixed in 3.7% paraformaldehyde for 30 minutes. For treatments of E8 intact retinas with PMA, retinas were isolated and incubated in DMEM without FCS in the presence of PMA as indicated in the figure. After incubation, retinas were lysed in electrophoresis buffer, or fixed in 3.7% paraformaldehyde for 30 minutes and processed for cryosection as previously described Fertilized hen eggs were kindly supplied by Prodhin Uruguay, and incubated in our laboratory at 37°C in a humidified atmosphere. For all procedures chick embryos were rapidly decapitated once extracted from the shell. E4 and E8 chick embryo neural retinas were excised and the cells isolated and cultured as described 152/156 from Sigma Aldrich, MO, USA); Martin D202-D87 . This was used to produce a rabbit anti-serum according to standard immunization protocols. Briefly, one rabbit was primed by multiple intradermal injections with a total of 500 µg of MARCKS protein in Freund’s adjuvant, followed by two 300 µg boosters by intramuscular injection at days 30 and 54. Bleeding to obtain the antiserum was at day 65. Recombinant protein was produced at the Recombinant Proteins Unit, Institut Pasteur de Montevideo, Uruguay (https://www.pasteur.edu.uy/upr) and the antibody at the Biotechnology Service, Polo Tecnológico de Pando, Universidad de la República, Uruguay (http://www.polotecnologico.fq.edu.uy/). Western-blot characterization of this antibody is shown in To generate the Polo52 rabbit polyclonal antibody, whole recombinant, His-tagged, chicken MARCKS protein was produced in bacteria. Briefly, MARCKS full coding sequence  and K39, substituted by arginine in medaka fish (O. latipes). S25, a site specifically phosphorylated in chick neuroblasts Nucleotide sequence information about MARCKS is available for five classes of vertebrates: mammals, birds, reptiles, amphibians and bony fishes. Therefore, it is possible to evaluate a representative number of primary structures. Indeed, a comparison of many of them . Figure Cell biology findings showed that MARCKS phosphorylation at S25 disappears in dissociated cells, but that it recovers as a function of time in culture, reaching a steady state one day after plating in vitro and treated with different kinase inhibitors during the last one or two hours, a time in which de novo MARCKS phosphorylation should be progressively increasing  and processed for immunoblotting with different antibodies. pED-MARCKS appeared evident at the end of the PMA pulse and its amount increased as a function of time, while S25p-MARCKS progressively decreased from 5 to 60 minutes . AltogetWe explored whether neuroblasts contain a Ser/Thr-protein phosphatase that stimulated by the PMA treatment could account for the observed S25p-MARCKS dephosphorylation. Many years ago it was shown by Yamamoto et al. 50, we assayed another compound known to be a more specific PP1 inhibitor. Tautomycetin selectively inhibits PP1 over PP2A The treatment with inhibitors with a higher specificity for PP2B, cyclosporin A and FK506 did not prevent, nor attenuate, MARCKS dephosphorylation at serine 25 . There wTaken together, our results indicate that MARCKS serine 25 is dephosphorylated by PP2A when the cells are stimulated with PMA.in vivo already at E4, although only the mitotic cells were immunoreactive. It must be remembered that at this developmental stage there is an important cell proliferation activity in the neural retina, which explains why this isoform constitutes a large fraction of the total protein. pED-MARCKS cell distribution has recently been described in dividing human hepatic stellate cells, where MARCKS was highly phosphorylated and localized at distinct sub-cellular regions in each one of the mitotic phases To analyze the relative amounts of two differently phosphorylated MARCKS variants present in the same immunoreactive electrophoretic band, we introduced here a new and simple procedure for processing Western-blot images. Merging digitally colored immunoreactive bands permitted us to obtain a quick semi-quantitative evaluation of polypeptide amounts. This procedure was used to compare the quantity of total MARCKS, pED-MARCKS and S25p-MARCKS during neural retina development, as well as in several different experimental situations. Therefore, it was possible to show that MARCKS was present since the earliest neural retina developmental stage assayed here, and that the same holds for S25p-MARCKS , confirming our earlier findings in vitro assays Taking into account the comparison of MARCKS protein sequences from several vertebrate species, a high homology was found in the region named “MH2”, S25 to E49 in the chick sequence (S26-E50 in humans) in vitroOur results from kinase inhibition experiments indicate that MARCKS phosphorylation at S25 is maintained by a permanent activity of a Cdk, which must be counteracted in cells by some unidentified phosphatase(s). As cell-cycle related Cdks are not active in differentiating retinal neurons in vivo, derives from the observation that both proteins are found accumulated in the same cells in the retina, and partially co-localize in growing neurites and growth cones. Their incomplete subcellular co-localization is most probably related to the fact that, in differentiating neurons, Cdk5 is associated to the cytoskeleton, where it phosphorylates many other substrates Additional information to reinforce the idea that Cdk5 does indeed interact with MARCKS 2) for 2 minutes. In these cells, the 80 kDa protein (MARCKS) was rapidly phosphorylated to a maximum followed by a decay attaining minimal values 6 minutes after PBt2 withdrawal We previously showed that the persistence of S25 phosphorylation depends on actin filaments stability It is known from the results of Yamamoto in vitro. In fact, Yamamoto et al. Most of the previous research regarding MARCKS serine dephosphorylation performed either using the purified protein or on different cell types, has been devoted to those residues located at the ED and phosphorylated by PKC. ED dephosphorylation has been attributed to phosphatases PP1A, PP2A, PP2B and PP2C From our experiments, PP1 and PP2B can be excluded as putative phosphatases for MARCKS serine 25, given the non-inhibitory effects of tautomycetin (PP1), FK506 and cyclosporine A (PP2B). Experiments using calyculin A as protein phosphatase inhibitor pointed to a form of PP2A as the most probable phosphatase involved in MARCKS S25 dephosphorylation. It is known that this enzyme is highly versatile depending of its regulatory subunit . Western-blot of E12 chick neural retina. A 0.2% Triton X-100 retina lysate containing 10 µg of protein was separated in 10% SDS-PAGE and sequentially immunodetected with anti-MARCKS polyclonal antibodies  and mAb 3C3. Observe that Polo52 antibody recognizes only one broad band at around 70 kDa. It behaves as the other anti-MARCKS antibodies MCt and Martin D202-D87.(TIF)Click here for additional data file.Figure S2Dissection and culture of differentiating neuroblasts from peripheral E4.5 chick retinas. Neuroephitelial cells were isolated form peripheral regions of E4.5 chick retinas. These dissociated cells differentiate in culture as retinal ganglion cells (RGCs) expressing the specific marker RA4 (A) Schematic representation of an extended E4.5 chick neural retina, showing the dissection procedure followed to obtain the retinal neuroepithelial cells. ONH, optic nerve head. (B) Percent of S25p-MARCKS- and RA4-positive cells as a function of time, in cultures from E4.5 peripheral neural retina cells. The dotted line represents the percent of cells with fragmented nuclei (in advanced stages of apoptosis). S25p-MARCKS and RA4 values were normalized to mean values at 2 hours after seeding. Values are represented as mean±SEM, n = 2 independent cultures, counting 1000–2000 cells in each. The ascending RA4 and S25p-MARCKS graphs show that at 24 hours most of the neurons differentiated in vitro. (C) General aspect of neuroepithelial cell cultures 24 hours after explantation. (D) Cultured neuroblasts exhibiting growing neurites 24 hours after explantation. Scale bars: C, 50 µm; D, 7 µm.(TIF)Click here for additional data file.Figure S3Flotation assays. Sequential Western-blots showing the presence of total MARCKS, S25p-MARCKS and Cdk5 in low-density membranes (rafts) obtained after sucrose-gradient centrifugation of a lysate from chick embryo brains (E12). The raft fraction is characterized by the enrichment of the 120 kDa N-CAM isoform.(TIF)Click here for additional data file.Table S1Assessment of cell death/suffering signs after a prolonged treatment of E4 peripheral retina cell cultures with the Cdk inhibitor roscovitine.(PDF)Click here for additional data file.Video S13D reconstruction of untreated E8 chick retina cell cultures. MARCKS is labeled in green and S25p-MARCKS in red. Note the presence of MARCKS protein in neuroblasts and Müller glia, while S25p-MARCKS is only present in neuroblasts. Both, MARCKS and S25p-MARCKS are localized in peripheral speckles in neuroblasts and almost every MARCKS cluster contains phosphorylation at S25.(MP4)Click here for additional data file.Video S23D reconstruction of E8 chick retina cell cultures treated with 300 nM PMA during 10 minutes. S25p-MARCKS is shown in red and pED-MARCKS in green. Note the reduction of S25p-MARCKS compared to the untreated situation Click here for additional data file.


The isolated physalins included the previously reported Hh inhibitors 5 and 6. Compounds 1 and 4 showed strong inhibition of GLI1 transcriptional activity, and exhibited cytotoxicity against cancer cell lines with an aberrant activation of Hh signaling. Compound 1 inhibited the production of the Hh-related proteins patched (PTCH) and BCL2. Analysis of the structures of different physalins showed that the left part of the physalins was important for Hh inhibitory activity. Interestingly, physalin H (1) disrupted GLI1 binding to its DNA binding domain, while the weak inhibitor physalin G (2) did not show inhibition of GLI1-DNA complex formation.Hedgehog (Hh) signaling plays an important role in embryonic development, cell maintenance and cell proliferation. Moreover, Hh signaling contributes to the growth of cancer cells. Physalins are highly oxidized natural products with a complex structure. Physalins ( Hedgehog (Hh) signaling plays an important role in embryonic development and adult tissue maintenance –2. Once 5; IC50, 0.66 μM) .–22.17–22, 38 μM) , caldeno0.45 μM) , taepeen 1.6 μM)  and a fl 0.5 μM)  were isoSolanum nigrum was identified as an active plant with our cell-based assay screening. After the removal of chlorophyll from the MeOH extract of Solanum nigrum (leaves) (7.9 g) by Diaion HP-20, each MeOH soluble part of fraction 1A (3.7 g) and fraction 1B (1.2 g) were suspended in H2O/MeOH 9:1. The suspension thus obtained was successively partitioned between hexane, EtOAc and n-BuOH. Consecutive steps of silica gel column chromatography, and reversed-phase high-performance liquid chromatography (HPLC) of the active EtOAc extracts of fraction 1A and 1B yielded physalin H (1) [2) [3) [4) [5) [6) [7) [Among our extract library, in H (1) , physali (1) [2) , physali [2) [3) , isophys [3) [4) , physali [4) [5)  and a mi [5) [6)  and phys [6) [7) . The str1) and isophysalin B (4) showed a dose-dependent inhibition of luciferase production  with a low cytotoxicity against assay cells (2) showed a weak Hh inhibition . Physalin K (3) did not show Hh inhibition at all, even at a concentration of 17.9 μM. Previously, we reported on the isolation and evaluation of Hh inhibitory activities of physalin F (5)  and physalin B (6)  [6) was isolated as a mixture with physalin C (7) at a ratio of 3:2. An equilibrium between 6 and 7 might exist under weakly acidic conditions, because the ratio of the mixture changed from 3:2 to 1:1 in a deuterated chloroform (CDCl3) solution after two months. Further, we examined the cytotoxicity against cancer cells, human pancreatic (PANC1) and prostate (DU145) cancer cell lines, in which Hh signaling is aberrantly activated (1)  showed cytotoxicity against PANC1  and DU145 . In addition, isophysalin B (4) and previously reported 5 and 6 also exhibited strong cytotoxicity against PANC1 . A moderate selectivity between differentiating cancer cells and normal cells was observed for active compounds 1, 4, 5 and 6, because the survival of C3H10T1/2 cells was not significantly affected by a treatment with these compounds. C3H10T1/2 is a normal cell line in which the Hh signalling pathway is active but does not contribute to abnormal cell survival. Interestingly, the right parts of these isolated physalins, except 7, were identical. Therefore, the difference in Hh inhibitory activity can be attributed to the left part of their structures. The difference in the Hh inhibitory activity between 4  and 6  indicates that an α,β-unsaturated ketone unit present in the physalin structure may be a good nucleophile acceptor and contribute to the Hh inhibition. In addition, a conjugated dienone system in 2 and a peroxy bridge in 3 may be associated with decreased Hh inhibitory activity. Western blot analyses were performed with physalin H (1), which was the most active compound, except previously reported compounds 5 and 6. Physalin H (1) showed an inhibition of Hh- related protein expression  is shown in 1) inhibited the expression of both proteins. In addition, at a concentration of 3.0 μM, physalin H (1) decreased the expression of BCL2 in DU145 cancer cells, in which Hh signaling was activated -conjugated streptavidin detected free “biotin-labeled GLI1-BS”  and inactive physalin G  was examined. Under these experimental conditions, physalin H (1) clearly inhibited the formation of GLI1–DNA complex at 200 μM. On the other hand, physalin G (2), which is similar in the right part of its structure to physalin H (1), did not inhibit the complex formation even at 200 μM. These results suggested that one of the Hh inhibitory mechanisms of physalin H (1) involves inhibition of GLI1–DNA-complex formation.Disruption of the GLI1–DNA complex in the final stage of this signaling pathway is one of the most attractive molecular mechanisms of Hh inhibitors. Recently, we reported the first small molecule, which directly inhibits the GLI1–DNA complex formation .GANT61, sequence ; Figure S. nigrum by using Hh inhibitory activity-guided isolation. Among them, four compounds  showed dose-dependent GLI1-transcriptional inhibitory activity. In addition, these physalins were found to be cytotoxic to cancer cells with an aberrant Hh signaling pathway and inhibited the expression of Hh signaling-related proteins. Structure–activity relationships were observed in the Hh inhibitory activity of physalins and the left part of the structure was found to have an important role in Hh inhibition. Moreover, physalin H (1) inhibited the formation of GLI1–DNA complex, but inactive physalin G (2) did not. To our knowledge, this is the second study in which the direct inhibition of the formation of the GL1–DNA complex by Hh inhibitors has been reported.We isolated seven physalins from The NMR measurements were carried out with JEOL ECP400 and ECP600 spectrometers in deuterated solvents and the residual proton chemical shift was taken as an internal standard. MS data were obtained with a JEOL JMS-T100LP (ESI). IR spectra were measured by using the ATR  method on a Jasco FT-IR 230 spectrophotometer. The DNA concentration was measured by a NanoDrop 2000 (Thermo Fisher Scientific), and the protein concentration was measured by an UVmini 1240 UV–vis Spectrophotometer . The procedures for the GLI1-mediated transcriptional activity assay, Western blot analysis and cytotoxicity test were as previously described . The GSTSolanum nigrum was collected in Bangladesh in 2011. A voucher specimen (KKB204) was deposited in the Ishibashi laboratory at the Chiba University.The plant S. nigrum (leave) (7.9 g) by Diaion HP-20, fraction 1A (3.7 g) was then partitioned between hexane, EtOAc and n-BuOH. The active EtOAc extract (623.4 mg) was subjected to silica gel column chromatography  to give 7 fractions (15A–15G). Fraction 15C (68.5 mg) was subjected to ODS HPLC  to give compound 2 , compound 3  and compound 1 . Fraction 1B (1.2 g) was partitioned between hexane, EtOAc and n-BuOH. The active EtOAc extract (294.5 mg) was subjected to silica gel column chromatography  to give 6 fractions (4A–4F). Fraction 4A (179.6 mg) was subjected to silicagel column chromatography  to give 5 fractions (5A–5E). Fraction 5C (50.7 mg) was subjected to ODS HPLC  to give 8 fractions (6A–6H). Fraction 6E (20.0 mg) was subjected to ODS HPLC  to give compound 5 . Fraction 5B (91.0 mg) was subjected to ODS HPLC  to give 6 fractions (7A–7F). Fraction 7E (11.4 mg) was subjected to ODS HPLC  to give compound 4 . Fraction 7D (34.0 mg) was subjected to ODS HPLC  to give a 2:3 mixture of compounds 6 and 7  and compound 4 . Fraction 4B was subjected to ODS HPLC  to give compound 3  and compound 1 .After the removal of chlorophyll from a MeOH extract of The double-stranded (ds) DNA fragments containing a GLI1 binding site (GLI1–BS) were prepared by annealing of single strand (ss) DNA; for biotin-labeled GLI1–BS, 5’-biotin-AGCTACCTGGGTGGTCTCTTCGA-3’ and 5’-biotin-TCGAAGAGACCACCCAGGTAGCT-3’. 10 μL of the ssDNA ) were mixed and annealed by PCR Thermal Cycler Dice  . For EMSA experiments, dsDNA TE buffer solutions were prepared as follows: biotin-labeled GLI1–BS (50 fmol/μL), unlabeled mutGLI1–BS and unlabeled GLI1–BS (5 pmol/μL). These solutions were stored below −20 °C.2, 4% glycerol) on ice . After incubation for 20 min on ice, protein–DNA complex, free DNA and free protein were separated on a 6% native polyacrylamide gel containing Tris-Borate-EDTA . After transfer of DNA onto a Biodyne B nylon membrane 0.45 µm  and DNA crosslinking by UV transilluminator , biotin-labeled DNA was detected by HRP-conjugated streptavidin, following the manufactures’ protocols.The assays were performed with a Light Shift Chemiluminescent EMSA Kit (Thermo). The binding reaction was set up as follows: 70 ng of GST–GLI1 protein, biotin-labeled GLI1–BS (20 fmol), and compound in DMSO  were combined in 2 μL of 10 × binding buffer (kit), (5 mM MgCl


Objective(s): Recently we reported that the soybean 15-lipoxygenase (SLO) inhibitory activity of pyrimidobenzothiazines largely depends on the orientation of sulfur atom of thiazine core towards FeIII-OH in the active site pocket of the enzyme with subsequent oxidation of sulfur to sulfoxide. In this paper the results of a comparative study on the SLO inhibitory activities of the mentioned compounds using ab initio calculations and docking analyses has been reported.Materials and Methods: Structure optimization and docking analyses were performed using HyperChem 7.5 and AutoDock Tools 4.0 respectively. Enzyme assessment was reduced using spectrophotometric MBTH-DMAB method. Results: The inhibitory activity of synthetic 2-substituted pyrimidobenzothiazines against soybean 15-lipoxygenase (SLO) was evaluated and structure activity relationships and binding modes of their 4-H and 4-methyl analogs were studied using docking analysis and ab initio calculations.Discussion: The results of these studies showed that the lack of 4-methyl substituent in the pyrimidobenzothiazine molecules greatly reduces their lipoxygenase inhibitory activities and it was also found that the HOMO energy difference between the 4-H and 4-Methyl analogs can be responsible for the observed inhibitory activity reduction.Conclusion: Our molecular modeling studies shows that by using more flexible amino acids during the docking process, more rational results can be obtained. The method of measuring the lipoxygenase activity is also of prime importance for the study of structure activity relationship. It is well documented that mammalian lipoxygenases (LO’s) are non-heme iron-containing enzymes responsible for the oxidation of polyunsaturated fatty acids and esters to hydroperoxy derivatives . These aEvidence for the inhibition of 15-LO in the treatment of vascular disease is, however, most compelling . Both trIt is also found that 15-LO is linked to cardiovascular complications since it is known to participate in oxidative modification of low-density lipoproteins (LDL) leading to the development of atherosclerosis .Three different strategies have been developed to inhibit the LO’s pathway . They inIII-OH in the active site pocket of the enzyme with subsequent oxidation of sulfur to sulfoxide  inhibitory activities of some pyrimidobenzothiazines derivatives 1a-f and 2a-f and on the basis of the structure activity relationship (SAR) studies we suggested that the inhibitory activity of these molecules largely depends on the orientation of sulfur atom of thiazine core towards chelated Feulfoxide , 15. In rotocols . In the rotocols .Chemistry1a-f and 2a-f were synthesized according to the procedure reported in the previous literatures by starting from uracil and 4-methyluracil as primary reagents   then the output files were minimized under ab initio methods with 6-311G* basis set . The initial guess of the MO coefficients is from eigenvectors of the core Hamiltonian in HyperChem 7.5  , 20. In  Chem7.5 . After gChem 7.5 .Crystal structure of soybean lipoxygenase-3 (arachidonic acid 15-lipoxygenase) complex with 13(S)-hydroproxy-9 (Z)-2,ll(E)-octadecadienoic acid was retrieved from RCSB Protein Data Bank (PDB entry: 1IK3). Molecular docking 1a-f and 2a-f into the active site of SLO with Auto Dock Tools version 4.2 (revision 30) (Automated docking simulation was implemented to dock sion 30)  using Lasion 30) . This mesion 30) , 24, 25.sion 30) .1a-f and 2a-f docked conformers corresponding to the lowest-energy structures. After docking procedure, docking results were submitted to DS visualize  while number of generations was set to 256. The 256 docked complexes were clustered with a root-mean-square deviation tolerance of 0.2 Å. The program generated 256 compound isualize  for furtSLO screening assay50 results are outlined in Linoleic acid and two assay solutions (A and B) were prepared in advance. Solution A was 50 mM DMAB in a l00 mM phosphate buffer (pH 7.0). Solution B was a mixture of l0 mM MBTH (3 mL), hemoglobin  in 50 mM phosphate buffer at pH 5.0 (25 mL). A linoleic acid solution was prepared by mixing 5 mg of linoleic acid with 0.5 mL ethanol and then diluting with KOH 100 mM to a final volume of 5 mL. In the standard assay, the sample in ethanol (25 µL), SLO  and phosphate buffer pH 7.0  were mixed in a test tube and preincubation was carried out for 5 min at room temperature. A control test was done with the same volume of ethanol. After the pre-incubation, linoleic acid solution (50 µL) was added to start the peroxidation reaction, and, 7 min later, solution A (270 µL) and then solution B (130 µL) was added to start the color formation. 5 min later, 200 µL of a 2% SDS solution was added to terminate the reaction. The absorbance at 598 nm was compared with control test. The IC3 hybridization, the more the bending; and because of its electron donating and reducing properties inhibitory role of the sulfur atom increases in thiazine ring. It has also been proved that in the process of the enzyme inhibition, the sulfur atom undergoes oxidation  and inhibitory constant (Ki) of each docked model was calculated and outputted as Kcal/mol and mol/L respectively  was chosen as “consensus structure” to study its structure-activity relationship  and DMAB (3-dimethylaminobenzoic acid) indicators in the presence of hemoglobin at the wavelength of 598 nm, the peroxide concentration was determined . The results showed that the IC50 of 1e and 2e increase to 129.2 and 39.2 µM while it was nearly unchanged for 1c and 2c which have had methyl piperidine moiety. This behavior originated from protonation decrease of N-methyl piperazine moieties at higher pH. Also it is seen that by changing N-methyl piperazine to N-ethyl piperazine (increase of steric hindrance) the IC50 value increases. For further proof, the bulker analog 2g with N-phenyl piperazine moiety and lower protonation potency was tested for SLO inhibitory activity. The IC50 value of 2g was 182 µM which could be another witness for the protonation assertion.Considering the data of Table 1 we can see among the two series of the synthetic compounds the lowest ICi) of the consensus structures versus the results obtained from the enzyme assay (IC50) was studied and is plotted graphically  for the hypothetical process:o = ∆Ho - T∆So ∆Go ~ 0 ΔGo = ∆Ho ∆Go = ∆EHOMO = -2.3RTlogK∆SHOMO/2.3RTlogK = -ΔEHOMO = EHOMO2 – EHOMO1ΔE5.374 - 4.805 = 0.5690) of the diagram in It is interesting that the average of the aforementioned calculated K (K = 0.655) is closed to Y-intercept difference  log IC50 - (4.334 ± 0.3163)log KThe results of our investigations regarding the previously mentioned derivatives show that any changes in HOMO energy state are as important as molecular docking results. For example, if we want to predict the inhibitory activity of a new derivative containing a bulkier substituent like ethyl group at position 4, besides obtaining the inhibitory constant through docking, we must also determine the difference between the HOMO energy states of this derivative with its 4-H analogs.Our molecular modeling studies shows that by using more flexible amino acids during the docking process, more rational results can be obtained. The method of measuring the lipoxygenase activity is also of prime importance for the study of structure activity relationship. The reason behind this argument is based on a comparison that we can make between the results of our previous and present investigations. In our previous study, the enzyme activity was measured by considering the formation of the diene at 234 nm, while we based our recent study on measuring the amount of the peroxide formed at 598 nm. In the previous method, due to the slight solubility of some derivatives and turbidity of their solutions, the error was marginally high in the range of UV wavelength, which had a negative effect on the results of enzyme activity. Furthermore, in the docking investigation, we found that amino acids in the active site of the enzyme were not flexible which reduce the chance of finding a suitable binding space for the inhibitor. Because of these two problems, in our previous investigation we did not get a suitable linear relationship between the results of molecular docking and of enzyme activity. We believe our recent investigation is more comprehensive than our previously reported study.


Evodia rutaecarpa Bentham (Rutaceae), reduces obesity and insulin resistance in obese/diabetic mice; however, the mechanism underlying the effect of evodiamine on insulin resistance is unknown. This study investigated the effect of evodiamine on signal transduction relating to insulin resistance using obese/diabetic KK-Ay mice and an in vitro adipocyte culture. There is a significant decrease in the mammalian target of rapamycin (mTOR) and ribosomal S6 protein kinase (S6K) signaling in white adipose tissue (WAT) in KK-Ay mice treated with evodiamine, in which glucose tolerance is improved. In addition, reduction of insulin receptor substrate 1 (IRS1) serine phosphorylation, an indicator of insulin resistance, was detected in their WAT, suggesting suppression of the negative feedback loop from S6K to IRS1. As well as the stimulation of IRS1 and Akt serine phosphorylation, insulin-stimulated phosphorylation of mTOR and S6K is time-dependent in 3T3-L1 adipocytes, whereas evodiamine does not affect their phosphorylation except for an inhibitory effect on mTOR phosphorylation. Moreover, evodiamine inhibits the insulin-stimulated phosphorylation of mTOR and S6K, leading to down-regulation of IRS1 serine phosphorylation in the adipocytes. Evodiamine also stimulates phosphorylation of AMP-activated protein kinase (AMPK), an important regulator of energy metabolism, which may cause down-regulation of mTOR signaling in adipocytes. A similar effect on AMPK, mTOR and IRS1 phosphorylation was found in adipocytes treated with rosiglitazone. These results suggest evodiamine improves glucose tolerance and prevents the progress of insulin resistance associated with obese/diabetic states, at least in part, through inhibition of mTOR-S6K signaling and IRS1 serine phosphorylation in adipocytes.Evodiamine, an alkaloid extracted from the dried unripe fruit of the tree  The increased availability of food in Western countries and in Japan has augmented the prevalence of obesity and insulin resistance, which are central in the development of metabolic syndrome Insulin signaling is implicated in the regulation of adipocyte biology. Many of the metabolic and anti-apoptotic effects of insulin are mediated by the signaling pathway beginning from phosphorylation and activation of insulin/insulin-like growth factor I receptors, which results in tyrosine phosphorylation of the insulin receptor substrate 1 (IRS1) Evodia rutaecarpa Bentham (Rutaceae), has been used for many years as a traditional Chinese herbal medicine for the treatment of pain, vomiting and pyresis. Evodiamine has a wide variety of bioactivity with antinociceptive, anti-obesity, vasodilatory, anti-tumor and anti-inflammatory effects in vitro to investigate how evodiamine affects glucose tolerance and insulin resistance, especially from the viewpoint of signal transduction, including the mTOR-S6K signaling pathway.Evodiamine, an alkaloid extracted from the dried unripe fruit of 2 and 3503±303 µm2; gonadal WAT (GWAT), 1845±205 µm2 and 1403±152 µm2; IWAT, 4112±592 µm2 and 3844±308 µm2 in the control and evodiamine group, respectively; We examined the effects of evodiamine on the metabolic phenotypes associated with obese/diabetic states in KK-Ay mice. When mice were treated daily with evodiamine or vehicle (control) for one week, a significant decrease in body weight (BW) gain, but not in food intake, was found in the evodiamine group compared to the control group . As showWe examined the effects of evodiamine on the signaling of mTOR and IRS1 in the obese/diabetic mice because attenuated phosphorylation of Akt, an upstream kinase for mTOR, is present in the WAT of the evodiamine-treated mice, as reported To assess the direct effects of evodiamine on adipocytes, we examined the effects of insulin and evodiamine on stimulation of the mTOR-S6K signaling pathway using differentiated 3T3-L1 adipocytes. As well as the stimulation of IRS1 and Akt phosphorylation, insulin-stimulated phosphorylation of mTOR and S6K was time-dependent; however, evodiamine had little effect on their phosphorylation . InsulinWe examined the effects of rosiglitazone, an anti-diabetic compound, on the phosphorylation of these signaling molecules compared to the effects of insulin and evodiamine in adipocytes. Similar to evodiamine, rosiglitazone alone did not affect phosphorylation of S6K, IRS1, Akt and ERK (except mTOR) in the cells but, like evodiamine, rosiglitazone suppressed insulin-stimulated phosphorylation of mTOR and IRS1 in adipocytes significantly , althougFrom the results in Evodiamine is a multipotent compound that has a wide variety of bioactivities, such as anti-obesity and anti-tumor effects in vitro. So, the contribution of decreased Akt phosphorylation in WATs to the regulation of glucose uptake may be little in the evodiamine-treated mice. Because phosphorylation of IRS1 Tyr612 and PDK1 Ser241 in the WAT was not changed by treatment with evodiamine .ad libitum. Eight-weeks old female mice were administered evodiamine by i.p. injection  or vehicle (10% (v/v) DMSO, 10% (v/v) Tween80, 80% (w/v) NaCl) once a day for 7 days. Compound solutions were kept at ∼35°C and injected i.p. at a dose of 100 µl/g BW (3 mg/ml). BW and food intake were recorded each day. After 1 week, ten mice for tissue analyses were euthanized by cervical dislocation; their blood and tissue samples were recovered and stored at −80°C. Total seventeen mice in two independent experiments for glucose tolerance test were fasted for 17 h after the final injection of evodiamine or vehicle.KK-Ay mice were obtained from CLEA Japan, Inc. and maintained at 23±1°C under artificial lighting for 12 h/day and provided a standard chow  and tap water Blood samples were collected from a tail vein and used immediately to determine the glucose level with a glucometer . Insulin levels were measured with serum and commercial assay kits . An i.p. glucose tolerance test (IPGTT) using 1.5 mg glucose/g BW was done after starvation for 17 h. The blood glucose level was measured before the injection of glucose at time zero and at 30, 60 and 120 min later.3T3-L1 cells  were grown in Dulbecco's modified Eagle medium  containing 10% (v/v) calf serum (BioWest). Adipocyte differentiation was done essentially as described but with minor modifications Western blot analysis was done as described with the total tissue lysates or whole-cell lysates recovered from the WAT or 3T3-L1 adipocytes Glucose uptake into 3T3-L1 adipocytes was evaluated according to the method of Saito et al. Immediately after removal, tissue pieces were fixed by immersion at 4°C in 10% (v/v) formaldehyde in neutral buffer solution , dehydrated, cleared and embedded in paraffin. Paraffin sections (4-µm thick) were stained with hematoxylin and eosin. All observations were done with a fluorescence microscope . The average cell size of adipocytes in WAT was calculated by dividing the chosen microscopic area by the total cell number in the area.p≤0.05.Data were expressed as mean±SEM. Differences between groups were assessed by analysis of variance (ANOVA) or repeated measure ANOVA. The level of statistically significant difference was set at File S1n = 5). *p<0.05 vs. vehicle group. Figure S2, Effect of evodiamine on phosphorylation of mTOR, Akt and IRS1 in the WAT of obese UCP1-KO mice. Western blot analysis for mTOR (A), Akt (B) and IRS1 (C) was done with tissue lysates (50 µg protein) of epididymal WAT from the UCP1-KO mice fed a high-fat diet with or without evodiamine for 2 months as described in Supplemental Methods. Phosphorylation levels of mTOR Ser2448, Akt Ser473 and IRS1 Ser636/639 were normalized to the total level of each protein. Data are expressed as mean±SEM (n = 4). *p<0.05 vs. control group. Figure S3, Effect of evodiamine on phosphorylation of mTOR and UCP1 in the BAT of KK-Ay mice treated with evodiamine. Western blot analysis for mTOR (A) and UCP1 (B) was done with tissue lysates (30 µg protein) of BAT from KK-Ay mice treated with evodiamine for 7 days. Levels of mTOR Ser2448 phosphorylation and UCP1 protein were normalized to the total mTOR and tubulin levels, respectively. Data are expressed as mean±SEM (n = 5). Figure S4, Effect of evodiamine on phosphorylation of mTOR, S6K, Akt and IRS1 in the gastrocnemius muscle (GM) of KK-Ay mice treated with evodiamine. Western blot analysis for mTOR (A), S6K (B), Akt (C) and IRS1 (D) was done with tissue lysates (30 µg protein) of GM from KK-Ay mice treated with evodiamine for 7 days. Phosphorylation levels of mTOR Ser2448, S6K Thr389, Akt Ser473 and IRS1 Ser636/639 were normalized to the total level of each protein. Data are expressed as mean±SEM (n = 5). Figure S5, Effect of evodiamine on phosphorylation of ERK, Akt and IRS1 in pre-adipocytes. 3T3-L1 pre-adipocytes were serum-deprived for 4 h and then treated with 20 µM evodiamine for 1 h and with 20 nM insulin for the last 10 min. Western blot analysis for ERK, Akt and IRS1 were done with cell lysates (30 µg protein). Figure S6, Measurement of oxygen consumption (VO2) and respiratory quotient (RQ) in UCP1-KO mice with or without treatment with evodiamine. VO2 (A) and RQ (B) were measured for 24 h in male mice injected with vehicle or evodiamine (3 mg/kg BW) at 14:00 h. C, Data on RQs were analyzed as relative cumulative frequency (PRCF). Each curve represents 1365 measurements of RQ from three mice for each group as described in Supplemental Methods. Data are expressed as mean±SEM (n = 3 for each group). ***p<0.001 vs. vehicle group.Methods S1, Animal experiments using UCP1-knockout (KO) mice and cell culture experiments using 3T3-L1 pre-adipocytes. References S1, References for supplementary methods. Figure S1, Effect of evodiamine on mTOR-S6K signaling in the RWAT of KK-Ay mice treated with evodiamine. Western blot analysis of mTOR (A), S6K (B), Akt (C) and IRS1 (D) was done with tissue lysates (30 µg protein) of retroperitoneal WAT from KK-Ay mice treated with evodiamine for 7 days. Phosphorylation levels of mTOR Ser2448, S6K Thr389, Akt Ser473 and IRS1 Ser636/639 were normalized to the total level of each protein. Data are expressed as mean±SEM ((DOCX)Click here for additional data file.


Plasmodium falciparum relies on a complex protein network that uses low and high affinity receptor–ligand interactions. Signal transduction through the action of specific kinases is a control mechanism for the orchestration of this process. In the present study we report on the phosphorylation of the CPD (cytoplasmic domain) of P. falciparum Rh2b (reticulocyte homologue protein 2b). First, we identified Ser3233 as the sole phospho-acceptor site in the CPD for in vitro phosphorylation by parasite extract. We provide several lines of evidence that this phosphorylation is mediated by PfCK2 : phosphorylation is cAMP independent, utilizes ATP as well as GTP as phosphate donors, is inhibited by heparin and tetrabromocinnamic acid, and is mediated by purified PfCK2. We raised a phospho-specific antibody and showed that Ser3233 phosphorylation occurs in the parasite prior to host cell egress. We analysed the spatiotemporal aspects of this phosphorylation using immunoprecipitated endogenous Rh2b and minigenes expressing the CPD either at the plasma or rhoptry membrane. Phosphorylation of Rh2b is not spatially restricted to either the plasma or rhoptry membrane and most probably occurs before Rh2b is translocated from the rhoptry neck to the plasma membrane.Red blood cell invasion by the malaria parasite  Plasmodium falciparum. Transmission of this protozoan pathogen occurs during feeding of an Anopheles mosquito. After an initial multiplication step in liver cells, the parasite invades erythrocytes and multiplies by a process known as schizogony within red blood cells. Rupture of a red blood cell marks the end of multiplication followed by re-invasion into new erythrocytes  from uninfected erythrocytes by magnetic sorting using VarioMACS (Militenyi Biotech). After saponin lysis, parasites were lysed in 10 volumes of ice-cold buffer B  by several passages through a needle and three cycles of freeze–thaw in liquid nitrogen. The membrane fraction was separated from the soluble proteins by centrifugation at 13000 g for 30 min at 4°C. Control extracts were made from similar amounts of uninfected red blood cells. Radioactive kinase assays were carried out in 35 μl of standard reactions containing kinase buffer , 25 μM ATP (Sigma) and 1–2 μg of recombinant fusion protein bound on glutathione–Sepharose beads (Genscript). Assays were carried out with or without 10 μM cAMP (Sigma). For kinase assays using recombinant MmPKAc  or ZmCK2α (Zea mays CK2 α subunit) (Biaffin), 300 units of kinase were mixed with the indicated amount of recombinant protein, kinase buffer and ATP on ice. Reactions were started with addition of 1 μCi [γ-32P]ATP  and incubated at 30°C for 40 min. Beads were washed three times with ice-cold buffer B and the reaction was terminated by the addition of 5× SDS loading dye with subsequent boiling at 95°C for 3 min. Proteins were separated by SDS/PAGE (15% gel) and stained with SimplyBlue™ Coomassie Safestain (Invitrogen). Finally, the stained gels were shrink-wrapped on 3MM Whatmann paper (Schleicher & Schuell) and autoradiograms were generated using RP NEW medical X-ray screens (CEA). Non-radioactive kinase assays were carried out as described above. Proteins were separated by SDS/PAGE (10% gel) and detected by Western blotting.Parasite lysate used in the For treatment with different inhibitors, heparin (Ratiopharm), TBCA  and staurosporine (Merck), samples were mixed with the indicated concentrations of inhibitory molecules and the treated and mock-treated samples were incubated for 5 min at 30°C, stored on ice and mixed with ATP. Reactions were started by incubation at 30°C for 40 min. GTP (Sigma) was used in the same concentration as ATP (25 μM) with the addition of 0.5 mM manganese chloride (New England Biolabs).An antibody recognizing the phosphorylated Rh2b CPD was raised against the phosphorylated Rh2b CPD peptide NNDHL(pS)NYADKE (3227–3239 aa) according to the manufacturer's protocols (Eurogentec). The antibody was purified in two steps. First, a column containing the phosphorylated peptide was used and all peptide antibodies were captured. In a second step, the specificity of mixed antibodies was improved by use of a column containing the unphosphorylated peptide with subsequent clearance of the flow through of non-specific IgG antibodies.For immunoblots, parasite proteins from a synchronized culture were separated on 7.5% (Bio-Rad Laboratories) or 10% SDS/PAGE and transferred on to nitrocellulose membranes (Schleicher & Schuell). Membranes were blocked with 6% skimmed milk and incubated in the first antibody overnight. Monoclonal anti-HA or monoclonal anti-GFP (green fluorescent protein) were used at a 1:1000 dilution (Roche), phospho-specific anti-pRh2b antibody was used at a 1:3000 dilution, polyclonal anti-BiP (immunoglobulin heavy-chain-binding protein) was used at a 1:2000 dilution  and polyA total of 120 ml of 8% synchronized Rh2b–HA parasites were harvested in the late schizont stage and the parasites were released from the host cells using saponin. Pure parasites were washed in ice-cold PBS and lysed in 50 mM Tris/HCl (pH 7.5), 150 mM NaCl, 0.5% NP-40 (Nonidet P40), Complete™ Protease inhibitor and Phosphatase Inhibitor Cocktail I. After separation of the membrane fraction by centrifugation, the supernatant was mixed with an anti-HA antibody (Roche) to a final concentration of 1 μg/ml and incubated with rolling at 4°C for 2 h. Protein G–agarose beads  were added and rolling continued overnight. Beads were then pelleted by centrifugation and washed three times in lysis buffer with two additional wash steps in lysis buffer without detergent. For phosphatase treatment, beads were split and one part was mixed with 400 units of Lambda PMP , whereas the other served as a control. Both were incubated for 15 min by 30°C, mixed with 5× SDS loading dye, boiled for 3 min at 95°C and separated by SDS/PAGE (7.5% gel) to perform Western blotting. For immunoprecipitation of CK2–HA, 40 ml of 8% mixed-stage parasites were released from the host cells by saponin and lysed as indicated above. After separating the soluble and the membrane fraction, the soluble lysate was pre-incubated with Protein G beads (Genscript) for 2 h, incubated with a final concentration of 1.5 μg/ml monoclonal anti-HA antibody for 3 h and finally the antigen–antibody complex was bound to Protein G beads overnight. After washing the antigen–antibody bead complex seven times in lysis buffer, beads were stored until use at −80°C. For the phosphorylation assays a volume of 5 μl of beads was used.IFAs of Rh2b–HA parasites were performed on fixed parasites using ice-cold acetone for 30 min. After rehydration [10 min PBS at room temperature (22°C)] parasites were incubated with anti-HA (1:500 dilution) antibody solution, washed and incubated in secondary goat anti-rat antibody coupled to Alexa Fluor™ 594 (1:2000 dilution) (Molecular Probes) together with DAPI . Parasites expressing CK2–HA were fixed using glutaraldehyde/formaldehyde , incubatin vitro phosphorylation assays with late-stage parasite lysate in the presence or absence of 10 μM cAMP. AMA1 was included as a control substrate. Whereas phosphorylation of recombinant GST–AMA1 was stimulated by cAMP, GST–Rh2b was already strongly phosphorylated in the absence of external cAMP. GST alone was not phosphorylated , but only two are predicted to be phosphorylated by NetPhos 2.0  [3213, as well as Ser3233 as an additional putative phospho-acceptor site, were targeted for further analyses.The CPD of Rh2b encodes four putative phospho-acceptor sites (Sere 0.961) . Owing te 0.961) ,32, onlyin vitro phosphorylation assays (3213 and Ser3233) resulted in a protein (Rh2bPhosphoMut) that is not susceptible for in vitro phosphorylation with parasite lysate, indicating that one or both of these residues are indeed targets for phosphorylation. Using single point mutations, Ser3233, but not the predicted Ser3213, was identified as the essential residue for this modification in vitro (wt(RBCcontrol), 3233 as a genuine phospho-acceptor site in vitro, point mutations substituting adjacent residues including Tyr3235 were generated . Although the role of CK2 in blood-stage parasite proliferation is well established [bona fide homologue for PKC could be identified in the Plasmodium genome [in vitro phosphorylation assays with parasite lysate. This polyanionic compound is known to inhibit CK2 of several organisms [http://www.biochemj.org/bj/452/bj4520457add.htm).The kinase prediction tool NetPhosK 1.0  predictsablished ,34, no bm genome , thus imm genome  and secom genome . In orderganisms . Owing trganisms ,39–41. TPfCK2 [2+ GTP and analysed for its phosphorylation status. Rh2b was found to be phosphorylated in the presence of ATP as well as in the presence of GTP, but not in the absence of a phosphate donor  a CK2-specific inhibitor, TBCA , was use 5–50 μM .50 value of 19 μM, whereas the enzyme's autophosphorylation is not affected by even a 32 μM concentration [T. gondii CK2-dependent phosphorylation is resistant to a 10 μM concentration of staurosporine using a Toxoplasma cell lysate [Rh2b phosphorylation by parasite lysate was significantly reduced by a TBCA concentration of 10 μM, whereas 50 μM TBCA completely abolished its modification C. These ntration . A previl lysate .In vitro Rh2b phosphorylation was not affected by 1 μM staurosporine, although cAMP-dependent AMA1 phosphorylation was efficiently blocked (T. gondii homologue. blocked D. A highhttp://www.biochemj.org/bj/452/bj4520457add.htm) or episomally overexpressed (CK2–HA) in 3D7 parasites  was either endogenously HA-tagged  phosphorylated Rh2b at Ser3233  was utiin vivo, a transgenic parasite line was generated expressing endogenous Rh2b with a C-terminal 3×HA tag  recognized two protein bands at 300 kDa and above  was the dominant form detected by the anti-pRh2b antibody, suggesting that the full-length Rh2b protein was significantly less phosphorylated E.in vivo was confirmed further by episomal overexpression of the two variants of the CPD as GFP-fusion proteins that allowed the differential targeting of the CPD to two specific membranes: the plasma and rhoptry membranes. In both membrane systems the CPD of Rh2b were phosphorylated in the late stages .The specificity of the anti-Rh2b antibody and Rh2b CPD phosphorylation in vivo absence. By contrast, phosphorylation prediction tools like NetPhos 2.0 do predict at least one phosphorylation site (above the arbitrarily implemented threshold of 0.5) within the analysed CPDs except for EBA175 and Rh1 .Previous studies on the CPD of AMA1 revealed specific phosphorylation mediated by PKA as one essential step during the invasion process ,22. Howe3233) in a cAMP-independent manner in in vitro assays. Its phosphorylation appears to correlate in vivo in late schizont stages mainly with N-terminal processing. A substantial line of evidence implicates PfCK2 in Rh2b phosphorylation: Ser3233 phosphorylation is sensitive to low amounts of heparin [PfCK2 as well as recombinant ZmCK2α phosphorylates Ser3233. CK2 is a highly conserved kinase that can be found ubiquitously in eukaryotic organisms [in vivo phosphorylation of PfGAP45  implicated CK2 activity in staurosporine-resistant phosphorylation [In the present study we show that the CPD of Rh2b is phosphorylated at a single residue . We found that the mutation S3233A in the CPD of a chimaeric Rh2a/b invasion ligand has no significant impact on the use of invasion pathways by D10 parasites . We therefore propose that phosphorylation of Ser3233 might trigger a pre-invasive process that could be connected to translocation of the Rh2b protein or to a putative role in signalling events in merozoites primed for invasion.The CK2-dependent phosphorylation of the CPD of Rh2b at Ser


Guanylyl cyclase C (GC-C) is a receptor expressed in intestinal epithelial cells and activation of GCC by its ligands elevates intracellular cGMP which results in an inhibition of cell proliferation . Multiplinvitro and in vivo assays, we show that GC-C is a substrate for phosphorylation by c-src. This phosphorylation now results in a reduction in guanylyl cyclase activity. Phosphorylation of GC-C by c-src allows the docking of the c-src SH2 domain to phosphorylated GC-C, which can now result in further activation of c-src. This feed forward mechanism of activation of c-src, induced by novel cross-talk between a receptor guanylyl cyclase and a tyrosine kinase, may have important implications in colonic tumour progression [We have investigated cross-talk of GC-C with additional signalling pathways in the intestinal cell in order to investigate its role in cell proliferation. Activation of c-src in human colonic cells results in down-regulation of cGMP production by GC-C, and through mutational analysis and gression . To idenThese observations may provide an explanation for the reduced incidence of colon-carcinoma seen in countries where the incidence of ST-mediated diarrhoea is higher. Moreover, our results also highlight the fact that the production of cGMP in intestinal epithelial cells is exquisitely regulated, and cGMP, in turn may regulate a number of pathways with diverse physiological effects.


Oral administration of these peptides to spontaneously hypertensive rats led to reductions in systolic blood pressure. In the present work, we examined the potential molecular mechanisms responsible for the ACE- and renin-inhibitory activities of these peptides. Enzyme inhibition kinetics showed competitive, non-competitive and mixed-type peptide-dependent inhibition of renin and ACE activities. Intrinsic fluorescence intensity data showed that LY and RALP have stronger binding effects on ACE molecule compared to that of TF. LY and RALP showed the highest inhibition of ACE and renin activities, respectively. Circular dichroism data showed that the inhibitory mechanism involved extensive peptide-dependent reductions in α-helix and β-sheet fractions of ACE and renin protein conformations. Molecular docking studies confirmed that the higher renin-inhibitory activity of RALP may be due to formation of several hydrogen bonds (H-bonds) with the enzyme’s active site residues. The rapeseed peptides inhibited renin and ACE activities mostly through binding to enzyme active site or non-active sites and forming extensive H-bonds that distorted the normal configuration required for catalysis. Data presented from this work could enhance development of highly potent antihypertensive natural peptides or peptidomimetics.Our previous study showed that three rapeseed protein-derived peptides  inhibited the  Renin and angiotensin-I converting enzyme (ACE) are the two key enzymes that regulate the renin-angiotensin system (RAS) and are important determinants of blood pressure and fluid homeostasis As an aspartyl protease, renin contains two catalytic aspartic acid residues (Asp32 and Asp215) that are located in the active site cleft and can accommodate seven amino acid units of the substrate (angiotensinogen). Renin’s catalytic activity involves cleavage of the peptide bond between Leu10 and Val11 of angiotensinogen to generate angiotensin-I in vitro inhibitions of renin and ACE activities The interest in bioactive peptides as agents for the control of hypertension continues to increase and our previous study has confirmed that rapeseed protein-derived peptides  possess dual The rapeseed protein-derived peptides Thr-Phe (TF), Leu-Tyr (LY) and Arg-Ala-Leu-Pro (RALP) were synthesized (>95% purity) by GenWay Biotech . Human recombinant renin protein  and renin inhibitor screening assay kit (10006270) were purchased from Cayman Chemicals . Rabbit lung ACE  and N-[3-(2-Furyl) acryloyl]-L-phenylalanyl-glycyl-glycine (FAPGG) were purchased from Sigma-Aldrich . Other analytical grade reagents were obtained from Fisher Scientific .maxV and mK) were calculated, respectively, as the Y and X-axis intercepts of the primary plot. iK was calculated as the X-axis intercept of the line obtained from a secondary plot of Lineweaver-Burk line slopes versus peptide concentrations.Kinetics of ACE and renin inhibition was determined using a previously described method Fluorescence emission spectra were obtained using the method of Li et al. Far-UV CD spectra were obtained using a JASCO J-815 spectropolarimeter  under constant nitrogen flush according to the method of Omoni and Aluko 2O-184 and H2O-250) were removed and hydrogen atoms added. A binding site with coordinates x: 7.568, y: 46.092, z: 68.842 and a radius of 10 Å was created around the ligand Molecular docking was performed using the Accelrys Discovery Studio software 2.5 (DS 2.5) according the method of Pan et al. p<0.05.All assays were conducted in triplicate and analyzed using one-way analysis of variance (ANOVA). The mean values were compared using Duncan’s multiple range test and significant differences accepted at 50 values of TF, LY and RALP were previously determined, respectively, to be 0.810, 0.107 and 0.648 mM for ACE inhibition, and 3.061, 1.868 and 0.968 mM for renin inhibition maxV (TF) and increased mK  values as shown in iK) is a measure of the peptide binding strength to ACE protein and a lower value usually indicates greater affinity. Therefore, 50 values. The iK values also seem to have a direct relationship with mK values as shown by the fact that the highest concentration-dependent increase in mK was associated with LY (88%) in comparison to RALP (42%). Thus LY binding to ACE protein was more effective in reducing catalytic activity when compared to the binding strength of RALP and TF.The ICmK values for TF are similar to that of the uninhibited reaction, which is typical of non-competitive inhibition. In contrast, the mK values for renin inhibition by LY and RALP are differed from that of the uninhibited reaction, which suggests a mixed-type mode of enzyme inhibition. The iK values suggest RALP is a more effective renin inhibitor when compared to LY and TF; the order of iK value is also positively related to peptide IC50 values. It was difficult to do a comparative analysis with previous works because there are almost no related renin inhibition kinetics studies with food protein-derived peptides in the literature. However, a previous work has shown that inhibition of in vitro renin activity by hempseed protein hydrolysates was mostly of the mixed-type The double reciprocal plot showed that TF acted as a non-competitive renin inhibitor  while LYTrp emission but with likely contributions from Tyr residues iK values as reported in Tyr and Trp) in ACE to the hydrophilic environment, which causes FI quenching. In addition to inducing an open protein structure, it is also possible that interaction of peptides with ACE led to shielding of previously exposed (in the native structure) Tyr and Trp residues, which will reduce the amount of light energy absorbed and hence reduced FI values. Since the wavelength of maximum FI (λmax) did not change (∼327 nm), it means that peptide interactions did not change the microenvironment of Tyr and Trp residues within the ACE protein structure. But the low value of 327 nm suggests that the Tyr and Trp residues were located in a mostly hydrophobic microenvironment within the ACE protein. The present results agree with a previous report that showed ostrich egg protein-derived peptides caused unfolding of ACE protein, which was accompanied by decreases in ACE FI Fluorescence emission spectroscopy can be used to evaluate the interaction and binding characteristics between peptides and enzyme proteins; information such as binding mechanism, binding constants and the number of binding sites may be obtained. Emission spectra of ACE in the presence of different peptide concentrations are shown in max from 328.5 nm in the absence of peptide to 326, 327 and 330 nm in the presence of 0.047, 0.094 and 0.188 mM peptide concentrations, respectively. Thus at 0.047 and 0.094 mM TF concentrations, there were slight increases in the hydrophobic environment of Tyr and Trp, which led to the blue shifts in λmax. However, at 0.170 mM peptide concentration, there seem to be a change in renin conformation into a more unfolded structure that modified the microenvironment of the Tyr and Trp residues to have a more hydrophilic character and hence an increase in λmax to 330 nm. For LY, only the 0.170 mM concentration produced a slight blue shift (326 nm) in λmax. In contrast to the observed effects of TF and LY on the microenvironment of Tyr and Trp in renin, RALP did not produce any concentration-dependent change in λmax.Peptide interactions with renin showed maximum FI at 328±0.5 nm when excited at 280 nm; the FI values were gradually decreased as peptide concentration was increased, which indicates modulation of renin protein conformation. However, the largest decrease (74%) in ACE FI was observed when LY was present at 0.170 mM concentration, which suggests greater affinity for renin protein when compared to smaller decreases in FI in the presence of TF (47%) or RALP (46%). As noted above, the decreases in renin FI in the presence of peptides indicate protein conformational changes that exposed hydrophobic groups to the hydrophilic environment. Interactions of TF with renin also led to concentration-dependent shifts in the λp>0.05) change in unordered structure. However, there were reductions in α-helix fraction while β-sheet and β-turn fractions were significantly (p<0.05) increased  in α-helix fraction. The most intense change caused by TF was observed at 0.094 mM, which increased the unordered fraction in renin protein structure by a factor of 2.25 while β-sheet fraction was reduced by a factor of ∼6. Similarly, the interactions of LY and RALP (each at respective 0.085 mM and 0.055 mM) with renin also led to significant (p<0.05) increases in unordered protein fraction  as well as decreases in content of β-sheet and α-helix  when compared to TF.The native renin protein also displayed a positive ellipticity band near 195 nm but only one negative band from 213 to 217 nm , which i α-helix . The res2+ showed the best poses (potE) of −31.36, −39.21, and −192.81 kJ/mol for TF, LY and RALP, respectively  on the helix α13 and Glu411 on the helix α14 of ACE protein 2+ account for the varied degrees of inhibition by peptides, and that the shorter the Zn2+ distance to the carbonyl oxygen of the peptide, the greater the degree of inhibition 2+ coordination distances with 7 µM and 18 µM IC50 values for ACE inhibition, respectively In addition to the hydrophobic and H-bond interactions, the interactions between ACE inhibitors and ZnArg may improve formation of multiple interactions and contribute to improved affinity of inhibitory peptides for renin. All the peptides were docked inside the active site of renin protein. For example, RALP occupied seven of the active site pockets  while TF and LY occupied five  and four pockets , respectively; these active sites have been previously described sp  of renin, which has been shown to be necessary for high affinity inhibition of enzyme activity sp and peptide modification to improve lipophilic character was suggested to be necessary for enhanced binding The docking results for peptide interactions with the active site of renin are shown in in vitro data clearly revealed that LY is a better inhibitor of ACE while RALP is a better renin inhibitor. The higher ACE-inhibitory activity of LY could be attributed to a higher collision rate constant and ability to convert the ACE protein into a more disordered structure. The mechanism responsible for the high renin-inhibitory activity of RALP probably involved the ability to form higher number of H-bonds with active site residues as well as the extensive reductions in α-helix and β-sheet fractions, which also resulted in a highly disordered enzyme protein structure. Results from this work also suggest that the presence of a positively-charged residue in RALP may have enhanced multiple interactions with negatively charged amino acid residues close to renin active site, which could have contributed to increased peptide inhibitory activity.In the present work, the interactions of TF, LY and RALP with ACE or renin have been successfully characterized using fluorescence spectra, CD spectra and molecular docking techniques in combination with enzyme inhibition kinetics. The Table S1Predicted binding energies .(DOC)Click here for additional data file.Table S2Hydrogen bonds observed between ACE (PDB: 1O86) or renin (PDB: 2V0Z) and the docked top ranked pose of peptides.(DOC)Click here for additional data file.Table S32+ coordination amino acid residues within ACE (PDB: 1O86) and bioactive peptides.The distance between Zn(DOC)Click here for additional data file.