Ascaris) are associated with increased allergy and this has been linked to cross-reactivity between worm proteins  and highly similar molecules in dust-mites and insects. The overlap between allergy and helminth infection is best illustrated in Anisakis simplex, a nematode that when consumed in under-cooked fish can be both an infective helminth and a food allergen. Nearly 20 molecular allergens have been isolated from this species, including tropomyosin (Ani s 3) and the EF-hand protein, Ani s troponin. In this review, we highlight aspects of the biology and biochemistry of helminths that may have influenced the evolution of the IgE response. We compare dominant IgE-antigens in worms with clinically important environmental allergens and suggest that arrays of such molecules will provide important information on anti-worm immunity as well as allergy.The Th2 immune response, culminating in eosinophilia and IgE production, is not only characteristic of allergy but also of infection by parasitic worms (helminths). Anti-parasite IgE has been associated with immunity against a range of helminth infections and many believe that IgE and its receptors evolved to help counter metazoan parasites. Allergens (IgE-antigens) are present in only a small minority of protein families and known IgE targets in helminths belong to these same families . During some helminth infection, especially with the well adapted hookworm, the Th2 response is moderated by parasite-expressed molecules. This has been associated with reduced allergy in helminth endemic areas and worm infection or products have been proposed as treatments for allergic conditions. However, some infections (especially  The parallels between allergy and the immune response to parasitic worms (helminths) have been noted for some time. Unlike most other inflammatory/infectious conditions, allergy, and helminths induce strongly Th2-skewed responses associated with cytokines such as IL-4, IL-5, and IL-13, with mastocytosis, eosinophilia, and antibody class-switching to produce IgE [reviewed in Ref.  above], it needs to be stressed that there are other immunity mechanisms operating as well; IgE is a late mammalian additional mechanism to the Th2-mediated mechanisms of lower vertebrates , thus IgE-immunity is not the only mechanism of immunity against metazoan parasites available to mammals. This is exemplified by the occurrence of Th2-like immune responses to helminth infection in avian hosts in the absence of IgE . FinallyTrichuris .Work in the allergy field has shown that very few protein families contain allergens  and impoOnchocerca volvulus . Tropomyosin is highly conserved across many invertebrates and is responsible for much of the IgE cross-reactivity between Ascaris and dust-mites  is an important IgE target in a number of nematode infections; volvulus , 77; Ascricoides ; AnisakiAni s 3, ]; and trst-mites . Cockroamolecule . SantiagSchistosoma japonicum  from invertebrate muscle targeted in IgE responses against aponicum , Ascarisricoides , Anisaki, anvolvulus . There iBlo t 11 . Cross-rBlo t 11 . Ani s 2 species .Ancylostoma Secreted Protein-2 (ASP-2), which was shown to be a potent IgE antigen in human studies in China and Brazil  has been recently shown to be an IgE antigen in mice  proteins are another family targeted by IgE. Hookworms secrete a VAL-like molecule, called d Brazil , 84. An d Brazil . ASP-2 bvolvulus , and Brua malayi , as well mansoni  and S. japonicum . Furtheraponicum . One of  in mice , but reqS. mansoni and an IgE response to some members of the TAL family has been associated with resistance to re-infection with the parasite  proteins are some of the most dominant IgE-antigens in parasite , 19, 22.oponin C  and the , FgCaBP .S-transferase (GST) is another source of IgE cross-reactivity. GST of nematode species is targeted by IgE during infection . These are large multimeric proteins that are cleaved into smaller fatty acid binding subunits  with funividuals  and thisividuals . The filividuals . Interesividuals .Sarcoptes scabiei). Interestingly, the IgE response to Sarcoptes scabiei is thought to be involved in protection against repeat infestation  have members in helminth species that are known to be targeted by IgE during infection, other common allergen families  have been identified in helminths, but their IgE binding has not yet been tested . Furtherestation .http://pfam.sanger.ac.uk/) of which only 255 have been identified in allergens . The debate about which functional and molecular properties make a protein an allergen has continued for some time (chitinases (AF041) have been described as panallergens in latex-fruit syndrome and are contained in a multitude of plants, such as Heveine [in latex,  deserves to be mentioned. Anisakis is the only currently known case of an organism being both a helminth parasite and a food allergen. The L3 larvae of the marine nematode A. simplex infect fish and cephalopods and consequently people that consume under-cooked seafood, however humans are a non-permissive host and the parasites cannot continue their life-cycle in man. Exposure to this helminth through food has been associated with allergic symptoms; asthma, rhinitis, dermatitis, and conjunctivitis , four bacterial, and three fungal species. Their analysis supported previous work by Emanuelsson and Spangfort , the IgE response to allergen-like SmTAL2 appears to be desensitized by the continuous exposure to small doses of the antigen, while the specific IgG4 response becomes pronounced  and one or more intermediate hosts. The life-cycle expression profile of allergen-like molecules influences the host response. For example, trematodes (flukes) such as schistosomes undergo asexual reproduction in snail species before releasing larvae that infect humans, which then develop into adult worms that produce eggs following sexual reproduction. Some of the schistosome allergen-like TAL proteins are developmentally transcribed . SmTAL1 –9 years . As indionounced . SmTAL6 onounced .Echinococcus granulosus (echinococcosis) have IgE to parasite antigens AgB, a protease inhibitor, Ag5, a serine protease, and EA21  and subsequent receptor cross-linking by parasitic allergens is needed for host protection. Is the activation of basophils, mast cells, and other IgE-bearing effector cells necessary for protection?It is well know that activation of mast cells and eosinophils can release proteases and toxic proteins , some of which have been shown to directly kill larval stages of parasites .de novo produced highly toxic polypeptides such as Granzyme B  associated with host protection? Do different IgE reactivity patterns correlate with various degrees of resistance to infection or post treatment re-infection?This is reminiscent of the situation previously encountered in allergy research, which relied on water-soluble extracts which are difficult to standardize for diagnostic purposes , and mayWhile the use of CRD in allergy diagnosis is conceptually slightly different , we suggest that a similar “molecule-based” approach would allow a better understanding of host resistance against helminths at the molecular level and, from a practical point of view, point the way to safer or more effective multi-target anti-helminthic vaccinations.If the IgE axis evolved to protect mammals against multi-cellular parasites, studying host responses to these organisms may teach us much about other IgE-mediated phenomena such as allergy. For example, characterizing parasite structures targeted by IgE may identify homologous molecules and potential allergens in novel foods and genetically modified organisms. The relationship between allergy and helminth infection brings costs and benefits. Elucidation of the molecular mechanisms by which some parasites moderate Th2 response in their hosts, may yield improved therapy for allergic conditions. On the other hand, treatment for the same worms in the developing world may inadvertently increase the prevalence of atopic disease. Moreover as a consequence of cross-reactivity between parasite and environmental allergens certain helminths can actually sensitize and aggravate allergy. Parasitic worm infections are a serious health problem in many countries and high resolution molecular techniques developed in the allergy field may help us to understand better the anti-parasite responses that are associated with immunity.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Fragilariopsis cylindrus, Chaetoceros simplex and Pseudo-nitzschia subcurvata) exhibited phenotypic plasticity in response to salinity and temperature regimes experienced during the seasonal formation and decay of sea ice. The degree of phenotypic plasticity, in terms of changes in macromolecular composition, was highly species-specific and consistent with each species’ known distribution and abundance throughout sea ice, meltwater and pelagic habitats, suggesting that phenotypic plasticity may have been selected for by the extreme variability of the polar marine environment. We argue that changes in diatom macromolecular composition and shifts in species dominance in response to a changing climate have the potential to alter nutrient and energy fluxes throughout the Southern Ocean ecosystem.Diatoms are the primary source of nutrition and energy for the Southern Ocean ecosystem. Microalgae, including diatoms, synthesise biological macromolecules such as lipids, proteins and carbohydrates for growth, reproduction and acclimation to prevailing environmental conditions. Here we show that three key species of Southern Ocean diatom ( Macromolecules, including proteins, lipids and carbohydrates are the building blocks of life. Microalgae are the primary source of macromolecules in marine ecosystems through their photosynthetic assimilation of dissolved inorganic carbon into organic carbon biomass that is then consumed by higher trophic levels. In the Antarctic, greater than 50% of this production is contributed by diatoms, which dominate the microalgal assemblage, ultimately providing food for krill, fish, whales, penguins, and seabirds . This caThe Southern Ocean is characterised by the seasonal formation and decay of sea ice, which produces a range of environmental conditions from deeply mixed waters in summer to solid ice sheets in winter ,7. ConsePhenotypic plasticity refers to an organisms’ ability to change their chemistry, physiology, development, morphology or behaviour, to maximise fitness in variable environments . PhotophLipid accumulation has frequently been reported in sea ice microalgal communities from both the Arctic and the Southern Ocean and is thought to provide an important source of nutrition and energy to higher trophic levels ,13. AlthDespite its importance, we have little understanding of macromolecular composition of individual microalgal species within natural assemblages. This is primarily due to technical limitations of traditional bulk analysis techniques, which do not have the capacity to target individual species within mixed communities. Fourier Transform Infrared (FTIR) microspectroscopy is a vibrational spectroscopic technique becoming increasingly popular amongst microbial ecologists ,18. The Fragilariopsis cylindrus, a dominant species in the sea ice and ice-edge habitats of both polar regions [Chaetoceros simplex, which is found in great abundance in pelagic and meltwater habitats around Antarctica and belongs to the most abundant algal genus on the planet [Pseudo-nitszchia subcurvata, typically found in meltwater habitats of coastal Antarctic waters [To improve our understanding of macromolecular plasticity and associated changes in nutritional value in response to salinity and temperature regimes characteristic of sea ice, meltwater and pelagic habitats, FTIR microspectroscopy was used to measure macromolecular concentrations in three Antarctic diatoms:  regions ; Chaetoce planet ; and Psec waters ,25. ThisF. cylindrus (Grunow) was collected from ice cores  taken in November 2001. Cultures of the Antarctic diatom C. simplex were isolated from the coastal waters of Antarctica, Prydz Bay  and P. subcurvata (Hasle) Fryxell was collected from the subpolar South Atlantic Ocean. No permission was necessary for the collection of the algal samples from the Southern Ocean, however permits were obtained from the Australian Quarantine and Inspection Service (AQIS) for the import of samples into Australia. The taxonomic classification of all species was confirmed, based on analysis of frustule morphology by light microscopy. All species were maintained in approximate pelagic conditions at 4°C in natural Antarctic filtered seawater , collected during SAZ-Sense, Jan.- Feb. 2007, RV Aurora Australis. Culturing was conducted in specially designed 1 L glass culturing vessels using natural seawater  enriched with F/2 nutrients, with continuous air bubbling and maintained at +4°C (Guillard and Ryther 1962). Cultures received a 16:8 h light:dark cycle at 50 µmol photons m-2 s-1 . Exponential growth phase (confirmed with regular in vivo auto-fluorescence measurements as a proxy for biomass [F. cylindrus) to acclimate before samples were removed and analysed [ biomass ; Trilogyanalysed . Pulse aApproximately 15 mL of cell suspension was filtered through 1 µm polycarbonate filter membranes using a hand-operated vacuum filter tower. Cells collected on the filter were then resuspended in isotonic saline solution  and MilliQ water) to wash the cells to remove F/2 medium which contains compounds that can absorb infrared radiation and possibly confound the FTIR measurements . The sal-1 with a Vertex 80v FTIR spectrometer  coupled with an IR microscope  with a Mercury Cadmium Telluride detector cooled with liquid nitrogen. The microscope was connected to a computer-controlled microscope stage and placed in a specially designed box which was purged with dehumidified air. The measurements were performed in the mapping mode, using a nominal aperture size of 5 µm × 5 µm with a spectral resolution of 8 cm-1, with 64 scans co-added. Adjusting the aperture for each species provided spectra that were representative unambiguously of individual cells (not clumps of cells) for C. simplex and P. subcurvata. However, for F. cylindrus spectra were acquired from groups of 5-10 cells due to a lower signal to noise ratio compared with the other species. The number of co-added scans was chosen as a good compromise between achieving spectra with good signal to noise characteristics and the rapid acquisition of data. Each treatment had four replicates and measurements were taken from a minimum of 50 cells per replicate. Spectra were processed using Happ-Genzel appodization and 2 levels of zero-filling. Spectral acquisition and instrument control was performed using Opus 6.5 software (Bruker).Spectral data were collected on the Infrared Microspectroscopy Beamline (2BM1B) at the Australian Synchrotron, Melbourne, Australia in August 2010 . Spectra were acquired over the measurement range 4000-800 cm-1 where spectra with maximum absorbance greater than 0.85, which resulted from spectral acquisition of regions of the sample where cells were clumped, were rejected. Spectra were then pre-processed taking the second derivative using the Savitzky-Golay algorithm with 9 smoothing points, and normalization using Extended Multiplicative Signal Correction (EMSC).FTIR spectral data was exported from the OPUS 6.5 for multivariate analysis using The Unscrambler X v 10.2 . An initial quality control procedure was performed over the range 3000-950 cmPartial Least Squares Discrimination Analysis (PLS-DA) modelling was used to classify samples by experimental treatment conditions and species based on their spectra using the Non-linear Iterative Partial Least Squares (NIPALS) algorithm on mean-centred data. Discriminant analysis based on the PLS approach is useful for dealing with complicated data sets where ordinary regression is difficult or impossible to apply . The NIP49,50. PLSDA used the spectral ranges containing bands of biological origin between 3050-2800 and 1780-1000 cm-1. Spectra with a predicted Y-value of ≥0.5 were accepted as being from cells of the relevant species or treatment; spectra with predicted Y-values ≤0.5 were rejected. To assess the validity (accuracy) of each model, sensitivity and specificity statistics were calculated using the equations True Positives/ and True Negatives/ respectively [For PLS-DA, infrared spectra from 200-300 individual cells was used to generate a PLS-DA model which was then validated using a test set of 200-300 individual cell spectra which were not used to generate the PLSDA models. The PLSDA training data matrix comprised the spectra (X-variables) and three Y variables with integer values of 0 or +1 coding for the three modelled spectral classes -1 from 200-300 individual cells (X-variables) in response to both temperature and salinity conditions (Y-variables). PLSR analyses were conducted on mean-centred data using the NIPALS algorithm and validated using an independent test set of 200-300 samples which were not used to generate the PLSR models.Partial Least Squares Regression (PLSR) analysis was used to compare variations in infrared spectra between the ranges 3050-2800 and 1780-1000 cmC. simplex samples using mass spectrometry based on the previously reported nitrogen to protein conversion factor for microalgae of 4.78 [C. simplex was previously reported to consist of 52% carbon [Relative changes in the concentration of lipids, carbohydrates, amino acids and phosphorylated molecules were estimated based on proportionality between absorbance and analyte concentration according to the Beer-Lambert Law, previously demonstrated with diatoms and other types of microalgal cells –33. Prot of 4.78 . Because% carbon . The calprotein is the mass of C from protein, Ctotal is the total mass of carbon and N is the mass of nitrogen per unit dry weight.Where CProtein measurements were then used as a training set to build a PLSR model based on 499 cell spectra. A further 295 cell spectra were used to validate the model. Experimental replicates that were used in the training data set were not used in the validation data set. This PLSR model was subsequently used to predict the protein carbon  for the remainder of the samples.Statistical analyses were conducted using SigmaPlot for Windows version 11.0 . If the data did not meet the assumption of homoscedasticity then a Kruskal Wallis on ranked data was used to replace the one-way ANOVA. In the case of a two-way ANOVA where data did not meet the assumption of homoscedasticity or normality, a non-parametric Scheirer-Ray-Hare test was used on ranked data instead. The Scheirer-Ray-Hare test was performed on Predictive Analytical Software  with additional calculations by hand and a Chi-squared table for determining the P-value. Given the reduced power of the S-R-H test, a more conservative significance level was set at α = 0.01 for these data. For all other analyses, significance level was set to α = 0.05.F. cylindrus, C. simplex and P. subcurvata) were exposed to three levels of salinity and temperature in order to simulate sea ice , meltwater  and pelagic  habitats. Changes in macromolecular composition were then measured using FTIR spectroscopy. Data are freely available from the authors upon request.Microalgal cells from each of the three species . This was indicative of a greater magnitude of change in macromolecular composition in F. cylindrus and C. simplex than P. subcurvata, as outlined below.The degree of plasticity was determined by comparing the accuracy of Partial Least Squares Discriminant Analysis (PLSDA) models used to classify “unknown” samples by treatment conditions. Samples which had a macromolecular composition more highly differentiated would be classified with a higher degree of accuracy than those which were more similar. F. cylindrus and C. simplex showed broad-scale changes in macromolecular composition including variation in levels of lipids (~1730 cm-1), proteins (~1650 cm-1), amino acids (~1400 cm-1) and phosphorylated molecules (~1250 cm-1), with a particularly strong increase in amino acid content evident for both species in the sea ice treatment . Additionally, the appearance of a second peak in the Amide I region at ~1630 cm-1 indicated that C. simplex underwent distinctive changes in protein composition . In contrast, changes in macromolecular composition in P. subcurvata were mainly restricted to proteins, which were at highest concentration in the pelagic and lowest in the sea ice treatment .Macromolecular composition was different between treatments for all three species, with variations in maximum absorbance for bands corresponding to biological macromolecules . F. cyliF. cylindrus a cluster of a few cells) and revealed more details about patterns of variation in macromolecular composition than could be determined from the average spectra , amino acids (1400 cm-1) and phosphorylated molecules (1260 cm-1) were higher in cells from the sea ice treatment compared to the meltwater or pelagic treatments, as shown in the average second derivative spectra . Additionally, a prominent and broad band at 1620 cm-1 indicted that changes in protein composition occurred between treatments. The Factor-2 loadings plot explained 19% of variation between cells and indicated that cells from the meltwater treatment had increased protein (1660-1540 cm-1) and carbohydrate (1040cm-1) content and decreased levels of phosphorylated molecules (1250cm-1) compared to the pelagic treated cells . Loadings plots for C. simplex showed fewer prominent peaks than for F. cylindrus, indicating that differences between cells were related to variations in fewer macromolecular pools. Factor-1 explained 34% of the variation between cells and was consistent with the average second derivative spectra , whereby cells from the sea ice treatment showed higher levels of lipids (1731 cm-1) and distinct changes in protein composition . Factor-2, which explained 28% of the variation between cells, showed that protein compositional changes occurred between meltwater and pelagic treatments . In the PLSDA scores plot for P. subcurvata, clustering of points by treatment was less distinct than for the other two species, indicating that much of the variability between cells was related to intra-population variation . The loadings plots for P. subcurvata were characterised by fewer prominent peaks than either of the other two species, confirming that changes were limited to fewer macromolecular pools . In combination, Factors-1 and 2 explained only 31% of variation between cells with moderate peaks at 1241, 1654, 1545 and 1020 cm-1 indicative of small variations in phosphorylated molecules, proteins and carbohydrates relative to the other two species. PLSDA scores plots provided a visualisation of clustering patterns in the data set, intrapopulation variability , suggesting a lower degree of plasticity in macromolecular composition in response to environmental changes in this organism.To further quantify the degree to which macromolecular composition varied with treatment for each species, Partial Least Squares Regression (PLSR) modelling was used to predict salinity and temperature levels based on cell spectra. A higher degree of accuracy indicated a more dissimilar macromolecular composition between treatments and hence a higher degree of plasticity for that species. Salinity and temperature could be predicted with accuracies of ± 12.1 and ± 2.6 °C, respectively, for  simplex . In contF. cylindrus showed the greatest magnitude of change, with lipid content nearly doubling in the sea ice treatment compared to the meltwater and pelagic treatments . C. simplex had the highest lipid content of the three species, regardless of treatment . Protein content varied between treatments with the pattern of variation being different for each species .Protein content was lowest in the sea ice treatment for all three species .. Sea ice conditions resulted in the lowest carbohydrate content for all three species . P. subcurvata had the highest and C. simplex the lowest carbohydrate content regardless of treatment . Amino acid content was significantly higher under sea ice conditions for both F. cylindrus and C. simplex  whereas P. subcurvata showed minimal variation in amino acid content. F. cylindrus demonstrated the greatest magnitude of change in amino acid content with levels more than doubling in the sea ice treatment compared to the meltwater and pelagic treatments. Phosphorylated molecules varied with both species and treatment . The lowest levels of phosphorylated molecules occurred in the sea ice treatment for F. cylindrus and the pelagic treatment for C. simplex, whereas P. subcurvata showed minimal variation between treatments.Lipid content increased under sea ice conditions in all three species . F. cylindrus and C. simplex, two diatom species with widespread distribution in diverse habitats in the Southern Ocean, each displayed a high degree of phenotypic plasticity, in terms of macromolecular composition, in response to changing temperature and salinity. In contrast, P. subcurvata, a meltwater specialist [P. subcurvata under sea ice treatment conditions provided further evidence that this species is less well adapted to the sea ice environment [F. cylindrus and C. simplex is advantageous under the highly variable physicochemical conditions associated with the seasonal formation and decay of sea ice.This study compared the phenotypic plasticity of three ecologically important species of Southern Ocean diatoms in response to salinity and temperature regimes characteristic of the highly variable polar marine environment. Photophysiological plasticity in response to these conditions was previously reported in Petrou et al. . This paecialist , displayironment . TogetheF. cylindrus and C. simplex in response to the sea ice treatment are consistent with previous observations of lipid accumulation by diatoms in the sea ice. P. subcurvata did not display a similar increase in lipid concentration.Microalgae vary macromolecular pools to satisfy physiological requirements. Lipids are used as storage products, for the repair and maintenance of membranes ,12,36–39C. simplex and F. cylindrus showed a distinct change in protein secondary structure (indicated by a shift in the Amide I peak maxima from 1650 to between 1630 and 1616 cm-1) attributed to a decrease in α-helix dominated proteins and an increase in proteins with β-pleated sheet. Antarctic microalgae have previously been reported to contain high amounts of Ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO), the enzyme which catalyses carboxylation and oxygenation of ribulose-1,5-bis- phosphate (RuBP) in the chloroplast [F. cylindrus and C. simplex (the two psychrophilic diatoms) cell spectra in the sea ice treatment is consistent with an increased proportion of RUBISCO in the protein pool. Cell spectra from P. subcurvata did not indicate similar changes in protein secondary structure.Protein concentration decreased in all three species in the sea ice treatment. In addition, oroplast . The autoroplast  the obseF. cylindrus and C. simplex showed elevated levels of free amino acids under sea ice treatment conditions, whereas P. subcurvata did not. Proline is known to be a key osmoregulator in Fragilaropsissp. and sea ice diatoms in general [F. cylindrus and C. simplex in study is consistent with previous research indicating that proline production is likely to be another important adaptive response to life in the sea ice habitat. Additionally, the catabolism of proline upon cessation of salt stress releases a great deal of energy which is thought to assist with the cells’ physiological recovery [Both  general . Hence, recovery .F. cylindrus and C. simplex, the stability of the meltwater habitat may have selected against phenotypic plasticity in P. subcurvata, resulting in a reduced capacity to vary macromolecular composition in response to environmental conditions, as shown in this study. For comparison, the extremophile alga Chlamydomonas raudensis UWO241 which was isolated from a permanently ice-covered lake has also demonstrated a high level of phenotypic plasticity[F. cylindrus and C. simplex, may have greater resilience to environmental fluctuations associated with global climate change than species such as P. subcurvata [Phenotypic plasticity can have significant ecological and genetic costs, which can reduce a species' fitness in comparison to a species with a less plastic phenotype . The coslasticity. Althouglasticity,48. Therbcurvata .Chaetoceros and Fragilariopsis species have already been observed in the Arctic growing under ice between 0.5 and 1.8 m thick in the Chukchi Sea [As the Antarctic region warms due to global climate change, polar inhabitants will experience warmer winters with thinning and retreat of sea ice cover . Such enkchi Sea . The autkchi Sea . Similarkchi Sea .Taxon-specific studies of microalgal physiology and associated effects on the nutritional value of natural populations are rare, particularly from the Southern Ocean ,54–56. SF. cylindrus and C. simplex, which belong to two of the most abundant genera in the Southern Ocean, lipid content was highest in cells from the sea ice treatment. Lipid accumulation has previously been reported in Antarctic sea ice algal communities due to the enhanced production of polyunsaturated fatty acids, resulting in a different quality of food being released into the food web in winter compared to other seasons [F. cylindrus and C. simplex was lower in treatment conditions characteristic of the sea ice habitat, years with reduced duration and extent of sea ice may be associated with increased protein production by sea ice microalgae. Given that microalgae are the major primary source of protein for the Southern Ocean food web, a macromolecule that is essential for growth at higher trophic levels [This study identified species-specific variations in cellular levels of all four major classes of macromolecules  in response to salinity and temperature regimes characteristic of sea ice, meltwater and pelagic habitats. For  seasons . Given t seasons . Howeverc levels , the relTable S1Infrared Band Assignments.(DOCX)Click here for additional data file.Table S2PLSDA Classification by Species Summary Statistics.(DOCX)Click here for additional data file.


Leishmania alternates between an invertebrate and a mammalian host. Upon their entry to mammalian macrophages, Leishmania promastigotes differentiate into amastigote forms within the harsh environment of the phagolysosomal compartment. Here, we provide evidence for the importance of translational control during the Leishmania differentiation process. We find that exposure of promastigotes to a combined elevated temperature and acidic pH stress, a key signal triggering amastigote differentiation, leads to a marked decrease in global translation initiation, which is associated with eIF2α phosphorylation. Interestingly, we show that amastigotes adapted to grow in a cell-free medium exhibit lower levels of protein synthesis in comparison to promastigotes, suggesting that amastigotes have to enter a slow growth state to adapt to the stressful conditions encountered inside macrophages. Reconversion of amastigotes back to promastigote growth results in upregulation of global translation and a decrease in eIF2α phosphorylation. In addition, we show that while general translation is reduced during amastigote differentiation, translation of amastigote-specific transcripts such as A2 is preferentially upregulated. We find that A2 developmental gene regulation is triggered by temperature changes in the environment and that occurs mainly at the level of translation. Upon elevated temperature, the A2 transcript is stabilized through its association with polyribosomes leading to high levels of translation. When temperature decreases during amastigote to promastigote differentiation, the A2 transcript is not longer associated with translating polyribosomes and is being gradually degraded. Overall, these findings contribute to our better understanding of the adaptive responses of Leishmania to stress during its development and highlight the importance of translational control in promastigote to amastigote differentiation and vice-versa.The parasitic protozoan  Leishmania spp. are the causative agents of leishmaniasis, the collective name for a number of important parasitic diseases affecting over 12 million people in 88 countries worldwide Leishmania undergoes marked morphological and biochemical transformations that are essential for its adaptation and survival to the rapidly changing environments in the macrophage Leishmania promastigote to amastigote differentiation in vitroLeishmania species are regulated at the level of individual mRNAs and up to 12%–18% are modulated at the protein level Leishmania donovani promastigote to amastigote differentiation in vitroL. donovani promastigotes and axenic amastigotes revealed important differences in protein phosphorylation profiles across the two major Leishmania lifestages Environmental stimuli like elevated temperature and low pH have been shown to be crucial in triggering Leishmania PERK eIF2α kinase homolog and addressed its role in the parasite’s differentiation within macrophages Leishmania amastigote differentiation process In eukaryotes, one of the best conserved stress response pathway is the phosphorylation of the alpha-subunit of eukaryotic initiation factor-2 (eIF2α) at serine 51 by stress-responsive kinases, resulting in the reduction of global translation Leishmania promastigotes exposed to a combined high temperature and low pH stress, a key signal triggering amastigote differentiation, exhibit a marked reduction in global translation, which coincides with eIF2α phosphorylation. Protein synthesis is also globally downregulated in axenic in vitro-generated amastigotes as compared to promastigotes while translation of known amastigote-specific genes is preferentially increased. Attenuation of global translation during amastigote differentiation may be important for allowing the parasite to adapt to the harsh environment of the phagolysosome in its mammalian host.Here, we report that Leishmania promastigote to amastigote differentiation within the phagolysosome of mammalian macrophages Leishmania adapts and survives within the harsh phagolysosomal compartment, we first investigated the effect of elevated temperature and low pH  on global translation using sucrose density gradient centrifugation to analyze ribosome profiles and metabolic cell labeling to evaluate protein synthesis rates. L. infantum promastigotes grown in RPMI medium at 25°C and pH 7.3 were exposed to either acidic or heat-shock stresses for different time points. Gradual exposure of L. infantum promastigotes for 3, 5 and 8 hours to an acidified medium or to a temperature shift from 25°C to 37°C had no significant effect on global translation, as determined by polysome profiling analysis  and monosomes (80S)  L. infantum promastigotes into amastigote-like forms in MAA-20 cell-free medium at 37°C and pH 5.5 for 4 days led to a significant decrease in global translation, as determined by ribosome profile analysis . Together these findings indicate that upon various stresses leading to a reduction in global translation, housekeeping transcripts such as alpha-tubulin are less efficiently translated.Under promastigote growth conditions , the olysomes . Howeverolysomes , consistA2 transcript markedly increased under heat stress. The A2 transcript is specifically expressed in amastigotes  or protein expression , but also in adapted amastigote cultures, suggesting that this life stage has to enter a slow growth state in order to adapt to the harsh environment in the phagolysosomal compartment. In addition, we show that while general translation is decreased during amastigote differentiation, translation of developmentally regulated transcripts like A2 is preferentially enhanced, in agreement with the induction of an amastigote-specific differentiation program.In this study, we explored the role of translational control in the context of Leishmania experiences an important number of stressful conditions, including rise in temperature, a dramatic shift in extracellular pH, increased levels of oxygen and nitrogen-reactive species, a high proteolytic activity and a nutritional stress within the phagolysosome. These external stimuli induce dynamic alterations in the regulation of gene expression, mainly at the posttranscriptional level L. infantum promastigotes to a combined elevated temperature and acidic pH stress for few hours (3–5 hrs) leads to a marked decrease in general translation. A similar decrease in polysome formation following the differentiation signal has been reported recently in L. donovani but after longer exposure times (e.g. 15–24 hrs) Leishmania’s development. Thus, the highly replicating promastigote forms undergo more active translation than intracellular amastigotes. These conclusions are consistent with previous reports demonstrating preferential upregulation in promastigotes of genes involved in translation  During its amastigote differentiation inside macrophages, Leishmania infective promastigotes experience stress upon their entry in macrophages and that translation attenuation mediated by eIF2α phosphorylation is important to allow the necessary adaptations for the parasite’s intracellular survival in the hostile environment of the phagolysosome. EIF2α phosphorylation is linked to amastigote differentiation as reconversion of amastigotes back to promastigote forms results in a dramatic decrease of eIF2α phosphorylation levels and upregulation of translation. Recent work from our laboratory highlighted the importance of eIF2α phosphorylation for amastigote differentiation as parasites unable to phosphorylate eIF2α by the ER resident PERK eIF2α kinase were markedly delayed in their differentiation into amastigote forms within macrophages Trypanosomatidae such as LeishmaniaTrypanosoma cruziApicomplexaToxoplasma gondii, it has been shown that eIF2α phosphorylation is important for maintaining latency of the bradyzoite form in the mammalian host Toxoplasma eIF2α is critical for the resistance of the parasite to external stress when outside its host and that inhibition of this process significantly delays the development of acute toxoplasmosis in vivoPlasmodium, phosphorylation of PfeIF2α by the IK2 kinase plays an important role in salivary gland sporozoite latency and transformation into liver stages Leishmania, interestingly in the related parasite Trypanosoma brucei, it has been shown that heat-shock stress up to 41°C causes a decrease in polysomes and the formation of stress granules but independently of eIF2α phosphorylation Eukaryotic cells have evolved mechanisms to respond to various cellular stresses encountered in their environments. One of the best-characterized stress response pathways conserved from yeast to humans is the reversible phosphorylation of the alpha-subunit of eukaryotic initiation factor 2, which lowers global protein synthesis along with induced translation of selected mRNAs Leishmania to conserve energy while reconfigurating the expression of specific sets of genes necessary for its survival in the mammalian host. Consistent with this hypothesis it has been shown previously that the effects of elevated temperature and/or acidic pH induce the expression of several individual mRNAs A2 amastigote-specific gene A2 transcript occurs mainly at the level of translation and that is triggered by elevated temperature and not by drop in the pH. Through its higher association with polyribosomes, the A2 transcript is stabilized and is more efficiently translated. When the temperature shifts from 37°C to 25°C during amastigote to promastigote differentiation, the A2 transcript is not longer associated with translating polyribosomes and is being gradually degraded. Our data indicate that although the A2 transcript could be detected for more than 24 hrs following amastigote to promastigote differentiation in vitro, A2 translation was significantly diminished already ∼5 hrs upon differentiation, suggesting that A2 downregulation during amastigote to promastigote switch is also controlled at the level of translation. It has been shown recently that the A2 protein is induced by heat shock (40°C) and that under these conditions is complexed with the ER chaperone BiP L. donovani to survive the higher temperatures associated with visceral organ infection. Despite its suggested localization in the ER following heat stress, our data indicate that A2 protein expression is not induced upon ER stress.Reduced translation during amastigote differentiation may allow Leishmania is capable of withstanding stressful situations during its digenetic life cycle by regulating general translation through reversible eIF2α phosphorylation. Global attenuation of translation during amastigote differentiation likely explains the slower growth rate of amastigotes inside the phagolysosome under conditions where metabolic requirements are reduced as compared to extracellular promastigotes A2 gene. Overall, these findings contribute to our better understanding of the adaptive responses of Leishmania to stress during its developmental switches and highlight the importance of translational control in promastigote to amastigote differentiation and vice-versa.In this study, the findings indicate that Leishmania infantum MHOM/MA/67/ITMAP-263 strain used in this study has been described elsewhere L. infantum promastigote to amastigote differentiation in a cell-free culture and the maintenance of axenic amastigotes were carried out as described previously 2 ventilated flasks and grown at 37°C and pH 5.5 with 5% CO2 for 5 days in average.The Leishmania lysates were clarified by centrifugation at 13,000×g for 15 min at 4o C. Protein quantification was assessed using the Bradford reagent (BioRad) and 25 µg of total protein lysates was loaded onto 10% SDS-PAGE. The gels were transferred on Immobilon-P polyvinylidene difluoride membranes (Millipore) following the manufacturer’s instructions. For the anti-alpha tubulin and anti-A2 antibodies, blocking was carried out for 60 min in phosphate-buffered saline with 5% non fat dry skim milk or 5% BSA, respectively prior to the addition of the first antibody. The A2 antibody was kindly provided by Dr Greg Matlashewski (McGill University). For the anti-eIF2alpha [pS51] phosphospecific antibody, blocking was performed O/N at 4°C in 5% BSA, prior to incubation for 2 hrs at RT° in 1% BSA. Mouse monoclonal antisera raised against L. infantum eIF2alpha Total RNA was isolated with Trizol™ (Invitrogen) following the manufacturer’s instructions. Northern blot hybridizations were performed following standard procedures 9L. infantum late-log/stationary-phase promastigotes treated with 0.4 µM or 1 µM thapsigargin or 5 µg/ml tunicamycin (Sigma) or subjected to heat-shock or acidic stress or to amastigote differentiation in culture were incubated with 100 µg/ml cycloheximide (Sigma) for 10 min, washed with cycloheximide-containing PBS buffer and lysed with a Dounce homogenizer in lysis buffer . Leishmania lysates were pelleted by centrifugation and the supernatant (40 OD260 nm units) was layered on top of a 15% to 45% linear sucrose gradient (10 ml) in gradient buffer  as described previously A total of 3×107L. infantum promastigotes grown in RPMI-1640 medium were subjected to either heat-shock from 25°C to 37°C or to low pH (from pH 7. 3 to pH 5.5) or to both conditions for different time periods. Thirty minutes before each time point, 0.5 ml of the culture was transferred to a methionine-free medium (Gibco) supplemented with 1 µCi/ml [35S]-methionine  and re-incubated under the same conditions for the rest of the stress period. Cells were pelleted, washed twice with ice-cold phosphate-buffer saline (PBS) and lysed in SDS-PAGE sample buffer. [35S]-methionine incorporation was measured with a scintillation counter (Beckman LS 6000TA). Samples were analyzed by SDS-PAGE and autoradiography.Approximately 5×10L. infantum promastigotes were washed twice in PBS and incubated in a methionine-free medium supplemented with 1 µCi/ml [35S]-methionine for 1 hour at 25°C. Following two washes in PBS, stress was induced in a cold methionine-containing medium (Gibco) at 37°C and pH 5.5. After different time points, 0.5 ml of culture were washed twice in PBS and the cells were lysed in Laemmli buffer. Non stressed cells were grown for the same period as the stressed cells and treated the same way. Values were normalized with the non-stressed control. [35S]-methionine incorporation was measured with a scintillation counter (Beckman LS 6000TA).Figure S1Global translation is reduced in L. major promastigotes subjected to temperature and acidic pH stress. Polysome profile analysis of L. major LV39 promastigotes (no stress) and parasites exposed to a combined stress of elevated temperature (37°C) and acidic pH (5.5) for 4 hours. Cell lysates were sedimented on 15% to 45% sucrose gradients. Gradients were fractionated and absorbance (Abs) at 254 nm was continuously recorded. The 40S and 60S subunits, 80S monosome and polysome peaks are indicated. Data displayed represent one of two separate experiments.(TIF)Click here for additional data file.Figure S2Morphological changes of L. infantum during axenic amastigote differentiation. Morphological analysis of L. infantum axenic differentiation from elongated flagellated promastigotes into round aflagellated amastigote-like forms using a phase-contrast microscope. L. infantum promastigotes (Pro) exposed to either elevated temperature (37°C) or a combination of high temperature and low pH (pH 5.5) for 4 and 8 hours, respectively are shown here.(TIF)Click here for additional data file.


We demonstrate that the WNK (WNK lysine-deficient protein kinase)-activated SPAK /OSR1 (oxidative stress-responsive kinase 1) known to directly phosphorylate and stimulate the N[K]CCs (Na+–K+ ion co-transporters), also promote inhibition of the KCCs (K+–Cl− co-transporters) by directly phosphorylating a recently described C-terminal threonine residue conserved in all KCC isoforms [Site-2 (Thr1048)]. First, we demonstrate that SPAK and OSR1, in the presence of the MO25 regulatory subunit, robustly phosphorylates all KCC isoforms at Site-2 in vitro. Secondly, STOCK1S-50699, a WNK pathway inhibitor, suppresses SPAK/OSR1 activation and KCC3A Site-2 phosphorylation with similar efficiency. Thirdly, in ES (embryonic stem) cells lacking SPAK/OSR1 activity, endogenous phosphorylation of KCC isoforms at Site-2 is abolished and these cells display elevated basal activity of 86Rb+ uptake that was not markedly stimulated further by hypotonic high K+ conditions, consistent with KCC3A activation. Fourthly, a tight correlation exists between SPAK/OSR1 activity and the magnitude of KCC3A Site-2 phosphorylation. Lastly, a Site-2 alanine KCC3A mutant preventing SPAK/OSR1 phosphorylation exhibits increased activity. We also observe that KCCs are directly phosphorylated by SPAK/OSR1, at a novel Site-3 (Thr5 in KCC1/KCC3 and Thr6 in KCC2/KCC4), and a previously recognized KCC3-specific residue, Site-4 (Ser96). These data demonstrate that the WNK-regulated SPAK/OSR1 kinases directly phosphorylate the N[K]CCs and KCCs, promoting their stimulation and inhibition respectively. Given these reciprocal actions with anticipated net effects of increasing Cl− influx, we propose that the targeting of WNK–SPAK/OSR1 with kinase inhibitors might be a novel potent strategy to enhance cellular Cl− extrusion, with potential implications for the therapeutic modulation of epithelial and neuronal ion transport in human disease states.Precise homoeostasis of the intracellular concentration of Cl − influx and efflux by reciprocally controlling the SLC12A family N[K]CC and KCC isoforms.WNK-regulated SPAK/OSR1 act as direct phosphorylators and major regulators of the KCC isoforms, which explains how activation of the WNK signalling pathway can co-ordinately regulate Cl Workthe KCCs . Hypotond Site-2 . Consistd Site-2 . The ided Site-2 . Consistd Site-2 ,40,43–45− influx and efflux by reciprocally controlling the SLC12 family N[K]CC and KCC isoforms. Given these reciprocal actions with anticipated net effects of increasing Cl− influx, we propose that inhibition of the WNK–SPAK/OSR1 signalling pathway might be a potent strategy to enhance cellular Cl− extrusion, with potential therapeutic implications.In the present study, we explore the mechanism by which WNKs might regulate KCC phosphorylation. Our data reveal for the first time that the WNK-controlled SPAK/OSR1 act as direct phosphorylators and major regulators of the KCC isoforms, and explain how activation of the WNK signalling pathway can co-ordinately regulate Cl96 antibody , anti-KCC3A phospho-Thr991 , anti-KCC3A phospho-Thr1048 WTKDKYM), anti-NKCC1 total antibody (residues 1–288 of human NKCC1), anti-NKCC1 phospho-Thr203/Thr207/Thr212 antibody , anti-WNK1 total antibody (residues 2360–2382 of human WNK1), anti-WNK1 phospho-Ser382 antibody ; anti-SPAK total antibody (full-length GST-tagged human SPAK protein), anti-SPAK/OSR1 (S-motif) phospho-Ser373/Ser325 antibody  and anti-ERK1  total antibody (full-length human ERK1 protein). The anti-FLAG antibody  was purchased from Sigma–Aldrich. Secondary antibodies coupled to HRP (horseradish peroxidase) used for immunoblotting were obtained from Pierce. IgG used in control immunoprecipitation experiments was affinity-purified from pre-immune serum using Protein G–Sepharose.The following antibodies were raised in sheep and affinity-purified on the appropriate antigen by the Division of Signal Transduction Therapy Unit at the University of Dundee: anti-KCC1 total antibody (residues 1–118 of human KCC1), anti-KCC2A total antibody (residues 1–119 of human KCC2A), anti-KCC3A total antibody (residues 1–175 of hu-man KCC3A), anti-KCC4 total antibody (residues 1–117 of human KCC4), anti-KCC3A phospho-Ser2, 1 mM MgCl2, 1 mM Na2HPO4, 1 mM Na2SO4 and 20 mM Hepes (pH 7.4). Hypotonic high potassium buffer was 80 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM Na2HPO4, 1 mM Na2SO4 and 20 mM Hepes (pH 7.4). Isotonic buffer was 135 mM NaCl, 5 mM KCl, 0.5 mM CaCl2, 0.5 mM MgCl2, 0.5 mM Na2HPO4, 0.5mM Na2SO4 and 15 mM Hepes (pH 7.5). Hypotonic low chloride buffer was 67.5 mM sodium gluconate, 2.5 mM potassium gluconate, 0.25 mM CaCl2, 0.25 mM MgCl2, 0.5 mM Na2HPO4, 0.5 mM Na2SO4 and 7.5 mM Hepes (pH 7.5).Buffer A contained 50 mM Tris/HCl (pH 7.5) and 0.1 mM EGTA. Lysis buffer was 50 mM Tris/HCl (pH 7.5), 1 mM EGTA, 1 mM EDTA, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1% (w/v) Nonidet P40, 0.27 M sucrose, 0.1% 2-mercaptoethanol and protease inhibitors (1 tablet per 50 ml). TTBS  was Tris/HCl (pH 7.5), 0.15 M NaCl and 0.2% Tween 20. SDS sample buffer was 1× NuPAGE LDS sample buffer (Invitrogen), containing 1% (v/v) 2-mercaptoethanol. Isotonic high potassium buffer was 95 mM NaCl, 50 mM KCl, 1 mM CaClhttp://www.dnaseq.co.uk).Full-length human KCCs isoforms were amplified employing SuperScript III (Invitrogen) from placenta total RNA (Stratagene) using the appropriate oligonucleotides. The RT–PCR (reverse transcription–PCR) products were ligated into the pCR2.1-TOPO vector and sequenced. The sequence-verified KCCs were subcloned into bacterial (pGEX-6P-1) and mammalian (pCMV5) expression vectors using BamHI and NotI. Required amino acid mutations were introduced into the pCR2.1-TOPO clone using site-directed mutagenesis by QuikChange method (Stratagene), but substituting the Taq enzyme with KOD Hot Start DNA polymerase (Novagen). Oligonucleotides were obtained from Invitrogen Life Sciences. DNA sequencing was performed by The Sequencing Service, College of Life Sciences, University of Dundee  and 5–10 μg of plasmid DNA as described previously  (0.5 μg) or kinase-inactive SPAK–SPAK [D212A] (0.5 μg) also purified from E. coli cells, at 30°C for 60 min in buffer A containing 10 mM MgCl2, 0.1 mM [γ32P]ATP (approximately 15000 c.p.m./pmol) in a total reaction volume of 25 μl. The reaction was terminated by the addition of LDS sample buffer. DTT was added to a final concentration of 10 mM, the samples heated at 95°C for 5 min and cooled for 20 min at room temperature. Iodoacetamide was then added to a final concentration of 50 mM and the samples left in the dark for 30 min at room temperature to alkylate the cysteine residues. The samples were subjected to electrophoresis on a Bis-Tris 10% polyacrylamide gel, which was stained with Colloidal Blue and then autoradiographed. The corresponding phosphorylated bands were excised, cut into smaller pieces and washed sequentially for 10 min on a vibrating platform with 1 ml of each of the following: water, 1:1 (v/v) mixture of water and acetonitrile, 0.1 M ammonium bicarbonate, 1:1 mixture of 0.1 M ammonium bicarbonate and acetonitrile, and finally acetonitrile. The gel pieces were dried before incubation for 16 h at 30°C in 25 mM triethylammonium bicarbonate containing 5 μg/ml trypsin as described previously ATP, and then digested them with trypsin. The resulting 32P-labelled tryptic peptides were isolated by HPLC and the identity and site of phosphorylation was determined by employing a combination of MS and solid-phase Edman sequencing  (96 (Site-4) within a motif not present in other KCCs  . Consisther KCCs . Our anaher KCCs . Importa− conditions  or hypotonic high K+ conditions (which should induce dephosphorylation of Site-1 and Site-2), to map further KCC3A phosphorylation sites in vivo [+ conditions did indeed induce a >10-fold reduction in phosphorylation of Site-1 (Thr991) and Site-2 (Thr1048)  . Our anaThr1048) . SeveralThr1048) .991), Site-2 (Thr1048) or Site-4 (Ser96) residues in KCC3A. The antibodies recognized by immunoblot or immunoprecipitation  and Site-2 (Thr1048) (+ conditions induced a significantly more moderate dephosphorylation of Site-4 (Ser96) compared with Site-1 and Site-2 (991), Site-2 (Thr1048) or Site-4 (Ser96) did not significantly affect the phosphorylation of the other sites  A. We obsd Site-2 A. Indivier sites A.+ conditions that induce the dephosphorylation of these residues.The residues surrounding Site-1 and Site-2 are almost identical in all KCC isoforms , indicatin vitro (− conditions that stimulate SPAK/OSR1 [382), SPAK/OSR1 phosphorylation at the residue phosphorylated by WNK1 (Ser373/Ser325) and NKCC1 phosphorylation at residues phosphorylated by SPAK/OSR1  over a 30 min time period , and then treated the cells in the same conditions with increasing doses of STOCK1S-50699 for an additional 30 min. These experiments confirmed that 1–10 μM STOCK1S-50699 inhibited markedly, in a dose-dependent manner, SPAK/OSR1 phosphorylation at Ser373/Ser325 (i.e. the activating site phosphorylated by WNK1) and NKCC1 phosphorylation at Thr203/Thr207/Thr212  in both hypotonic low Cl− conditions, as well as basic control isotonic conditions (1048) and Site-4 (Ser96) phosphorylation, consistent with the phosphorylation of these residues being mediated by SPAK/OSR1. Phosphorylation of Site-1 was also inhibited by STOCK1S-50699, but to a lower extent than Site-2  [96) [5 in KCC1/KCC3 and Thr6 in KCC2A/KCC4) that is also phosphorylated directly by SPAK/OSR1  . We also3A) [96) . Althoug3A) [96) , recent 3A) [96) . In addiPAK/OSR1 . Togethein vitro (1048) [Our findings indicate that the MO25α subunit is required for efficient phosphorylation of KCC isoforms by SPAK/OSR1, at least in vitro . This iso (1048) . Similaro (1048) ,40,43–45o (1048) , is cong991 in KCC3A) regulatory residue on KCC isoforms. The strongest and most direct evidence to support this conclusion is the finding that three endogenous KCC isoforms  are still phosphorylated on Site-1, but not on Site-2, in knockin ES cells that lack SPAK/OSR1 activity .Interestingly, our data suggest that SPAK/OSR1 may not phosphorylate directly the important Site-1 , we observed a stimulation of phosphorylation of Site-1 and Site-2 of the KCCs, closely accompanied with increased phosphorylation of WNK1, SPAK/OSR1 and NKCC1 (+) resulted in dephosphorylation of Site-1 and Site-2 in the KCCs, as well as WNK1, SPAK/OSR1 and NKCC1  and their associated regulatory subunits that tether these enzymes to their targets are likely to play a critical role in this pathway, illustrated by the fact that all KCC isoforms, as well as core WNK pathway components, are robustly dephosphorylated within 15 min of exposure of cells to hypotonic high K+ conditions  and also phosphorylate and inhibit K+-driven Cl− efflux co-transporters , provides a simple mechanism to explain how these critical Na+ and K+ co-transporters are reciprocally and co-ordinately controlled  in a more co-ordinated and balanced manner than thiazide and loop diuretics, which only suppress the activity of NCC (thiazide) or NKCC2 (loop diuretics) individually. Moreover, given the recent interest and proof of concept of small molecule enhancers of neuronal Cl− extrusion via KCC2 activation to treat diseases featuring GABAergic (GABA is γ-aminobutyric acid) disinhibition secondary to impaired KCC2 activity [− extrusion would be to concurrently inhibit Cl− influx via NKCC1 and activate Cl− efflux via KCC2 [The importance of co-ordinating cellular Clreciated ,17. The ntrolled . Stimuliactivity ,56, we pvia KCC2 ; our res


We also show that the transposition activity of MOS1 is downregulated by protein kinase AMP cyclic-dependent phosphorylation at S170, which renders the transposase unable to promote Mos1 transposition. One step in the transposition cycle, the assembly of the paired-end complex, is specifically inhibited. At the cellular level, we provide evidence that phosphorylation at S170 prevents the active transport of the transposase into the nucleus. Our data suggest that protein kinase AMP cyclic-dependent phosphorylation may play a double role in the early stages of genome invasion by mariner elements.Genomic plasticity mediated by transposable elements can have a dramatic impact on genome integrity. To minimize its genotoxic effects, it is tightly regulated either by intrinsic mechanisms (linked to the element itself) or by host-mediated mechanisms. Using mass spectrometry, we show here for the first time that MOS1, the transposase driving the mobility of the  The transposition of genetic elements is potentially genotoxic for the host cell despite being an essential tool for perpetuating genome plasticity, and thus for its evolutionary potential. This means that tight regulation of transposition is essential to maintain the balance between maintaining active transposons in host genomes and preventing the damage they could cause by potentially lethal DNA rearrangements. Studies of the relationships between transposable elements and the host genome have revealed diverse examples of the regulations that can be achieved through intrinsic, emergent or host-mediated mechanisms. Intrinsic regulation can be due to topological constraints , to the P-element of Drosophila  phosphorylation, a key reversible modification used as a regulatory mechanism in virtually every process in eukaryotic cells , and ii. While uosophila . The posscanning , but hasMos1 enters a naive genome. We found that MOS1 was phosphorylated at two residues: S2 and S170. The kinase responsible for S2 phosphorylation has not yet been identified, whereas S170 is strongly phosphorylated by the protein kinase AMP cyclic-dependent (PKA). Using biochemical approaches, we investigated the role of S2 and S170 phosphorylation (pS170) in MOS1 activity. The S2 phosphorylation has little or no effect. In contrast, the pS170 produces a dramatic decrease in MOS1 activity, which becomes unable to promote the transposition of a pseudo-Mos1 element in vitro. We identified the mechanism by which pS170 prevents transposition, and showed that it impairs the assembly of the paired-end complex (PEC), probably by steric hindrance and/or charge repulsion. In addition, we found that the cellular distribution pattern of pS170 MOS1 was different from that of the wild-type protein.To address this issue, we applied mass spectrometry (MS) methods to a MOS1 protein produced by insect cells, in a cell context free of transposition events. We did this to detect basal regulatory pathways, which could account for the inbuilt regulation of MOS1 when Sf21 cells. The fusion protein was purified onto a maltose binding resin  as described previously . His-MBP-MOS1 was similarly produced and purified from crude extract by affinity chromatography on a HiTrap column , and then on an amylose resin (NEB). The purified His-MBP-MOS1 was the kind gift of Guillaume Carpentier. For Xa-factor digestion, 5 µg of His-MBP-MOS1 or of MBP-MOS1 were incubated for 2 h at 25°C in 15 µl with 2 mM CaCl2, 5 mg of bovine serum albumin  and 1 µg of Xa-factor protease (NEB). The samples were boiled and loaded onto discontinuous 4–10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and then stained with ProQ Diamond (Invitrogen) or Coomassie blue. The Pro-Q Diamond phosphoprotein gel stain provides a method for selectively staining phosphoproteins in polyacrylamide gels, according to the manufacturer’s recommendations.MBP-MOS1 was produced using the pVL1392 baculovirus transfer vector and the BaculoGoldTM baculovirus expression system (BD Biosciences), and then extracted from baculovirus-infected eviously . After pEscherichia coli as described previously (Supplementary Table S1). MBP-S2D was produced and purified, as was MBP-MOS1.MBP-MOS1, MBP-S170A and MBP-S170D were produced and purified from eviously . MBP-S2DSf21-expressed MBP-MOS1 (10 µg) was incubated at 30°C for 45 min, with or without 15 U/µl lambda phosphatase (Sigma), in the recommended buffer consisting of 50 mM Tris/HCl, pH 7.5, 2 mM MnCl2, 5 mM dithiothreitol (DTT) and 0.1 mM Na2EDTA. The samples were acidified with formic acid at a final concentration of 1% (v/v) before desalting with a C4 ZipTip from Millipore . Desalted samples were analyzed by direct infusion in a 4-GHz MaXis ultra-high-resolution Q-TOF mass spectrometer from Bruker Daltonics (Germany) equipped with an electrospray ion source. Acquisitions were carried out in positive ion MS mode over a 300–3000 m/z range, with a nebulizer gas pressure of 0.3 bars. The drying gas flow and temperature were 4 L/min and 180°C, respectively. The acquisition rate was 1 Hz corresponding to spectra summations of 5494. External calibration was performed with ESI-L Low Concentration Tuning Mix (Agilent Technologies). ElectroSpray Ionization - High Resolution Mass Spectrometry (ESI-HRMS) spectra were processed and charge-deconvoluted using DataAnalysis 3.1 software  and the MaxEnt algorithm.m/z 100 to 2000 for MS, and from m/z 100 to 3000 for MS/MS. The MS acquisitions were carried out at enhanced resolution (8100 m/z units per second). Collision-induced dissociation (CIP) and electron-transfer dissociation (EDT) fragmentations were performed in separate nanoLC-IT-MS/MS runs.Cysteine reduction/carbamidomethylation was performed on MBP-MOS1 by a 30-min treatment at 56°C with a final concentration of 1 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) in 50 mM ammonium bicarbonate buffer, followed by 30 min alkylation at room temperature in the dark with 5 mM iodoacetamide. Alkylated MBP-MOS1 was cleaved for 4 h at 37°C with porcine trypsin at a 1:20 w:w trypsin:protein ratio. The tryptic phosphopeptides were enriched from the cleavage products by IMAC using iron-coated PHOS-Select™ metal chelate beads from Sigma in accordance with the manufacturer's instructions. Bound peptides were eluted using 1% phosphoric acid and analyzed by nanoLC-nanoESI-IT-MS/MS. Experiments were performed on the UltiMate™ 3000 NanoRSLC System  connected to a Bruker HCT Ultra PTM Discovery ion trap equipped with an online nanoelectrospray ion source. The LC-MS setup was controlled by the Bruker HyStar™ software version 3.2. Phosphopeptides were preconcentrated online on a Dionex Acclaim Pepmap100, 100 µm × 2 cm, C18 5 µm 100 Å reverse-phase precolumn and separated on a nanoscale Acclaim Pepmap100, 75 µm × 15 cm, C18 2 µm 100 Å column at a flow rate of 200 nl/min using a 5–45% gradient of acetonitrile in 0.1% formic acid. Mass spectra were acquired in positive ion mode from E. coli) were incubated with 170 units of cAMP-dependent protein kinase A catalytic subunit  and either γ32P ATP (25 µCi) or ATP (3.5 mM) in 40 mM Tris HCl, pH 7.4, and 20 mM Mg acetate, for 15 min at 30°C. Samples were then desalted on a protein desalting spin column (PIERCE). MBP5 (NEB) phosphorylation was achieved in a similar fashion. Radiolabeled proteins were analyzed on a 4–10% discontinuous SDS-PAGE. The gel was fixed, dried and exposed for 48 h on a Phosphor Screen (STORM Molecular Dynamics). For experiments to compare phosphorylated and nonphosphorylated MBP-MOS1 (from E. coli), the nonphosphorylated protein was subjected to the same procedure in the absence of PKA. They were designated +PKA and −PKA, respectively. Similar treatments were used to obtain phosphorylated MBP-S170A.Seventy-five micrograms of purified MBP-MOS1 , and 600 ng of supercoiled pBC-3T3 in a volume of 20 µl. The reactions were allowed to proceed for 30 min at 30°C, before 10 µl of stop solution  was added. The reaction mixtures were then incubated at 37°C for a further 30 min, and then at 65°C for 10 min. The products were phenol-chloroform extracted, and ethanol precipitated with 1 µg of yeast tRNA using standard techniques. Ten percent of the reaction mixture was co-transformed in 45 µl of electro-competent JM109 E. coli. Bacteria were grown at 37°C for 1 h in super optimal Broth (SOC) media. Appropriate dilutions of each reaction mixture were plated on LB-tetracycline (12.5 µg/ml) agar and Luria Broth (LB)-chloramphenicol (80 µg/ml) agar to score for transposition frequency. The transposition frequency was the number of TetR colonies divided by the number of ChloramR colonies + the number of TetR colonies.escribed . Basic t2, 0.5 mM EDTA, 0.5 mM DTT, 100 ng of BSA, 600 ng of supercoiled pBC-3T3 and 80 nM MBP-MOS1 from E. coli (±PKA as specified), in a volume of 20 µl. The reactions were allowed to proceed at 30°C for various periods (as indicated), before adding 2 µl of stop solution (loading buffer). The reaction mixtures were then incubated at 65°C for 10 min. Each reaction was analyzed by overnight electrophoresis at 2.7 V/cm on a Tris-Acetate-EDTA (TAE)-buffered 1% agarose gel. After electrophoresis, the gel was stained with ethidium bromide (0.3 mg/ml), and photographed on a transilluminator.Basic excision reaction mixtures contained 10 mM Tris-HCl (pH 9), 50 mM NaCl, 20 mM MgCl32P-labeled double-stranded oligonucleotide with the Mos1 3′inverted terminal repeat (ITR) sequence, and purified MBP-MOS1 from E. coli (±PKA as specified). To detect single-end complexes (SEC2), 0.2 pmol of uncleaved 3′ITR (UC-ITR) with 10 pmol of purified MBP-MOS1 were used , and reSupplementary Table S1). pCS2, in which the fusion proteins are expressed under the control of a pCMV  promoter, was used to prevent the overproduction of the fusion proteins, which could have altered the MOS1-GFP signal (Schneider-2 (S2) cells were transfected with the pCS2 expression vector containing either the green fluorescent protein (GFP) alone or the GFP in C-terminal fusion with the wild-type MOS1, or the S170A and the S170D mutants. The pCS2-GFP and pCS2-MOS1-GFP plasmids were a kind gift from Marie-Véronique Demattei . pCS2-S1P signal .2HPO4). The 2, and incubated at room temperature (RT) for 30 min. The 2+ suspension was added to 0.5 × 106 cells seeded in concanavalin-A–coated plates, and incubated for 24 h. Cells were washed, resuspended in media containing 1% FCS/700 µM CuSO4 and incubated for 72 h. The medium was removed from the plates, which were then washed with phosphate buffered saline (PBS).S2 cells were handled according to the Supplier's instructions (Invitrogene) and maintained at 26°C in Schneider-2 media with 10% (v/v) heat-inactivated fetal calf serul (FCS) (GIBCO) and 1.0 mg/ml PSG . Transfections were done using calcium phosphate. Briefly, 1 µg of DNA was mixed with 1× HBS buffer (21 mM hydroxyethyl piperazineethanesulfonic acid (HEPES), pH 7.1, 137 mM NaCl, 5.5 mM Dextrose, 50 mM KCl, 0.7 mM NaCells were fixed in PBS/2% paraformaldehyde at RT for 15 min, and then permeabilized with PBS/1% (w/v) Triton-X100 for 10 min. Hoechst 33342 dye was added at 1/250 and the cells then incubated for 30 min. Cells were washed with PBS and mounted for imaging. Single focal plates (1 μ) were acquired in a Leica DM5000B microscope, a CSU10 (Yokogawa) spinning disk head, a CoolSNAP HQ2 (Photometrics) CDD camera and the software MetaMorph . No postacquiring imaging processing was done except for adjusting the overall contrast of the images.mariner elements is now a well-established process, which involves several steps: (i) sequence-specific binding of a transposase dimer to the ITR present at one transposon end (forming a SEC2); (ii) pairing of the transposon ITRs to form a PEC; (iii) cleavage of both DNA strands at each transposon end; and (iv) integration of the transposon at a new locus. The Mos1 transposase (MOS1) is a 345-AA protein containing two domains: an N-terminal domain, which promotes the assembly of MOS1 dimers and their subsequent binding to Mos1 ITR  system, as this involves the expression of MOS1 in a cellular context similar to that of the native host (Drosophila species). Moreover, this expression system is efficient for phosphorylation and displays a high level of protein production. MOS1 was N-terminally fused with maltose binding protein (MBP). The MBP-tag was used both to achieve high-quality purification and to enhance MOS1 stability (Sf21 extracts was incubated with or without lambda (λ) phosphatase. Samples were analyzed by direct infusion ESI in an ultra-high-resolution Q-TOF mass spectrometer. Spectra from the untreated sample reveal four main peaks with masses of 83 307 (A1), 83 347 (A2), 83 386 (A3) and 83 426 Da (A4), respectively  could therefore be due to acetylation. The mass profile suggests that about half of the MBP-MOS1 analyzed was produced as an acetylated protein. Furthermore, a mass difference of +49 Da was measured between the theoretical mass of MBP-MOS1  and the observed mass (A1 = 83 307 Da). This mass difference could correspond to a point mutation (N to Y or H to W) or to extensive oxidation of methionine and/or tryptophan residues.Sf21 cells or E. coli were analyzed by SDS-PAGE, and stained with ProQ Diamond (Invitrogen) and Coomassie blue. The ProQ Diamond dye is used to detect phosphorylated proteins. As expected, ProQ Diamond staining revealed a signal for MBP-MOS1 produced from Sf21 cells, but not from bacteria  and 41 kDa (MOS1) were observed. Coomassie blue staining revealed that MOS1 was present as a less intense band than His-MBP, whereas ProQ Diamond labeled the MOS1 band much more intensively than His-MBP (sf21 cells was treated with phosphatases 1 or 2A (serine and threonine dephosphorylation), λ phosphatase  or LAR phosphatase (for tyrosine dephosphorylation). Our data indicated that only serines and threonines could be involved in MOS1 phosphorylation . No kinase was predicted as capable of phosphorylating S2, whereas S170 could be phosphorylated by at least 10 different kinases. Among these, PKA had the best prediction score, and the sequence surrounding S170 (165-PKRKKSYV-172) perfectly overlapped the PKA consensus phosphorylation site (RxxT/S). To corroborate these analyses, we performed in vitro phosphorylation of MBP-MOS1 produced in bacteria. Bacterially expressed MBP-MOS1 and MBP5 (NEB) were used in equal concentrations as substrates for PKA in the presence of γ32P ATP. After electrophoresis, MOS1 was confirmed to be efficiently phosphorylated by PKA treatment, since MBP-MOS1 was strongly labeled during the assay, whereas MBP was not. MBP-S170A  was treated in a similar way, and displayed drastically reduced protein labeling by PKA  (Mos1 (3T3) transposition with a rate of 10−4  reflected a decrease in specific activity as a result of treatment. The involvement of the S170 residue in MOS1 regulation by PKA phosphorylation was similarly analyzed. In vitro transposition assays were performed using the E. coli purified MBP-S170A mutant, treated with or without PKA. S170A treated by PKA was still able to promote the 3T3 transposition with a rate of 10−6, similar to that obtained without PKA. Another confirmation was obtained using the MBP-S170D mutant, a pseudo-phosphorylated MOS1 mutant that is unable to promote the 3T3 transposition. These data confirm a strong relationship between MOS1 transposition regulation by PKA and the S170 residue.The relationship between PKA phosphorylation and MOS1 activity was then investigated. osition) . The ass of 10−4 . We founMos1 transposition cycle are controlled by MOS1 phosphorylation, focusing first on single-end and PEC assembly. Electrophoretic mobility shift assays (EMSAs) were performed with MBP-MOS1 (±PKA) and the Mos1 3′ITR as a probe, using conditions appropriate for the detection of either SEC2 or PECs, as previously described (2 and 30°C)  is consistent with the data reported above. Hence, our data support the conclusion that the phosphorylation of the MOS1 S170 residue by PKA controls MOS1 activity by preventing PEC assembly.We went on to identify which stage(s) of escribed . SEC2 asnd 30°C) C. No sigdetected : first, Mos1 transposition cycle, PEC assembly precedes the first and second strand cleavages that promote excision , and the pBC-3T3 as a transposon donor. Our data show that MOS1 phosphorylated by PKA is unable to promote excision, as shown by the continued presence of the supercoiled donor, and the absence of backbone release even after incubating for 24 h . Taken together, these findings confirm that the phosphorylation of MOS1 by PKA prevents Mos1 transposition by inhibiting the assembly of PECs, and consequently further DNA cleavages.In the excision . We therfor 24 h . The prensposase . Under sIn vitro genetic ‘hop’ experiments were done using either purified MBP-MOS1 transposase or the MBP-S2D mutant and the pBC-3T3 plasmid, as the transposon donor and the target plasmid. Our data show that the S2D mutation has no effect on the transposase activity since the MBP-S2D promotes the pseudo-Mos1 (3T3) transposition with a rate of 7.10−5 .As previously indicated, the kinase responsible for S2 phosphorylation is unknown, making it difficult to perform the same studies as that performed for p170. Nevertheless, the use of pseudo-phosphorylated residues (D for pS) was demonstrated to be convenient (see our data with S170D), we prepare the S2D mutant. We assume that this mutant may help to describe the consequences of S2 residue phosphorylation. et al. have proposed that the nuclear localization of MOS1 is dependent on the presence of both an SV40 NLS-like motif (position: AA 168 to 174), and a dimerization subdomain located within the first amino terminal helix-turn-helix motif of the protein  was investigated by transient expression of plasmids coding for the full-length transposases fused at their C-terminal end with GFP , which locks the PEC by interacting with the linker region (residues 113–125) of the second monomer  plays a key role in regulating MOS1 activity, since the pS170 transposase is unable to promote transposition. This effect relies on the inability of the phosphorylated transposase to form the PEC and thus the subsequent transposition steps. On the other hand, pS170 does not impair the ability of MOS1 to form SEC2. By extension, this finding also shows that pS170 does not alter MOS1 dimerization. The crystal structure of the  monomer . More spP-element transposase may be down-regulated by ATM-kinase phosphorylation. Until recently, this was the only evidence of a phosphorylation being directly involved in regulating a recombinase activity. A novel component of the DSBs repair pathway, SETMAR, has recently been identified. SETMAR is a SET (histone methylase) and a mariner transposase fusion protein found only in anthropoid primates (SYV-172) does not overlap a Chk1 consensus phosphorylation site (LxRxxS/T). In contrast, the sequence surrounding S495 in SETMAR (489-YDNRRRSA-496) overlaps a PKA consensus phosphorylation site (RxxT/S), suggesting that SETMAR is not only sensitive to Chk1 regulation but could also be controlled by PKA. More generally, a PKA phosphorylation consensus is found around S170 for all the transposases of the mauritiana subfamily (to which MOS1 belongs), and also for the mariner transposases of the cecropia (to which SETMAR belongs) and mellifera/capitata subfamilies . In contrast, the mariner transposases of the irritans subfamily (to which HIMAR1 belongs) do not contain a PKA phosphorylation consensus site in the vicinity of S170.During the past decade, the relationships between eukaryotic DDE enzymes  activity and kinase activity have been addressed. The rationale underlying these studies is that the DDE recombinases cause DNA double-strand breaks (DSBs), which must be repaired if the cell is to survive. These repairs involve specific kinases [the ataxia telangiectasia mutated (ATM) or DNA-dependent protein kinases (DNA-PK)], and their involvement in regulating recombinases activity has been studied. Beall and colleagues  have shoprimates ,24. Receprimates . SETMAR primates . Interesmauritiana subfamily suggests that pS170 regulation plays a crucial role within the subfamily, whatever the host species. In contrast to Chk1 or the ATM and DNA-PK kinases, PKA is a cytoplasmic kinase and is the primary mediator of cAMP action in eukaryotic cells. As a consequence, biological conditions that activate PKA will concomitantly decrease the activity of MOS1 (and of the related transposases), before it can promote transposition and trigger DNA damage. PKA is implicated in diverse pathways that regulate cell survival and apoptosis in response to metabolic and growth signals, and it can elicit opposite effects depending on the cell type and situation. In addition to these activities, PKA has been shown to be involved in the infectious cycle of several viruses , thus preventing any major amplification of the element. This might be the case for Mos1 elements that are present in low copy numbers in their host-genome (up to 30)    and of r genome) . Hence, Supplementary Data are available at NAR Online, including .Francois Rabelais University (Tours - France); the Centre National de la Recherche Scientifique (CNRS); the French Ministère de l’Education Nationale, de la Recherche et de la Technologie; the Association Francaise contre les Myopathies [AFM # 11468 to CAG]; the Agence Nationale de la Recherche ; Région Centre/FEDER grant  to the CBM-ICOA Federation (in part toward high-resolution spectrometry). Funding for open access charge: ANR project (Elegineer).Conflict of interest statement. None declared.


Alu elements are active non-LTR retrotransposons that account for approximately 11 % of the human genome and are known to be not only the cause of genetic disease in germinal cells, but have also been implicated on the development of B-cell lymphomas suggesting they affect the genome of somatic cells as well. In the present study, the molecular mechanisms of the inhibitory activity of human APOBEC3 (A3) family members on Alu retrotransposition were investigated.r)-based retrotransposition assay. Briefly, HeLa cells were cotransfected with a neor-Alu reporter vector, LINE-1 ORF2 vector and the respective A3 expression plasmids. Seventy-two hours post-transfection, G418 was added for resistance selection, and after 14 days resultant G418-resistant colonies were fixed, stained with crystal violet and counted. A series of APOBEC3G (A3G) mutants bearing deletions in the N-terminus in multiples of 30 amino acids (aa) were constructed to evaluate the responsible region. Likewise, dimerization and deamination deficient A3G mutants were also created to examine the inhibitory effect of A3G on Alu retrotransposition.A3 family proteins were tested in a neomycin-resistant (neor-based retrotransposition assay showed that all hA3 family proteins differentially inhibited Alu retrotransposition. By the deletion analyses based on A3G that is the most well-characterized A3 family member, the N-terminal 30 aa of A3G was found to determine its inhibitory activity on Alu retrotransposition. Mutational analyses showed that the inhibitory activity of A3G was independent of its deaminase activity, but dependent on its dimerization, which was found to be affected by deletion in its N-terminal 30 aa. Importantly, the dimerization of A3G was also required for the inhibition of LINE-1 retrotransposition.The neoThe N-terminal 30 aa of A3G are essential for its inhibitory activity on Alu retrotransposition, and this correlates with A3G homodimerization. The structural basis for the effect of the N-terminal 30 aa region of A3G on its dimerization is currently under investigation.


Background and Aim. Calcineurin inhibitors (CNIs) have shown to develop hypertension in transplant patients. The in vitro incubation effects of cyclosporine (CsA) and tacrolimus (Tac) on vascular relaxations of rat thoracic aorta (TA) and abdominal aorta (AA) need to be investigated. Methods. The optimal concentrations of CsA (1.0 mg/mL) and Tac (0.1 mg/mL) used to compare endothelium-dependent (acetylcholine (ACh)) and endothelium-independent (sodium nitroprusside (SNP)) vascular relaxation against the agonists in phenylephrine (PE-) constricted TA and AA of 13-week-old male Sprague Dawley rats (n = 6). Results. In TA, the maximal vasodilator response elicited by ACh (control: Imax 98%) was significantly (P < 0.01) inhibited by CsA (Imax 10%) but not by Tac (Imax 97%). In AA,  CsA  showed strong sensitivity to inhibit ACh-dependent vascular relaxation than Tac (IC50 215 nM (P < 0.05); Imax 98%). CsA and Tac failed to affect the inhibitory responses to SNP in both TA and AA. Conclusion. CsA exerts profound inhibitory effect on endothelium-dependent vasodilatation as compared to Tac in both TA and AA. Aortic rings from the thoracic region are more sensitive to CNIs, since the vasodilator response to ACh is solely mediated by NO while in the AA, ACh likely recruits other endothelial mediators besides NO to maintain vasodilatation.  The results with the CNIs on the regulation of vascular function are indeed very conflicting owing to differences in study protocol, drug doses and treatment durations [The post-transplantation vasculopathy is an important complication after organ transplantation . The iniic rings . Howeverurations –11. To tin vitro incubation for a limited and short time of exposure for a 30 min duration with optimal and clinically relevant concentrations of either CsA (1 mg/mL) or Tac (0.1 mg/mL) would affect endothelium-mediated vasodilatation evoked by ACh and/or endothelium-independent vasodilatation induced by a direct VSM relaxing agonist such as SNP in phenylephrine (PE)-constricted rat aortic rings. This is the first report on the comparison of the differential and acute effects of in vitro addition of both CsA and Tac on endothelium-intact TA versus AA of rat aortic rings. In an attempt to address the above, the major goal of the present study is to examine whether acute The studies were performed using 13-week-old male Sprague-Dawley rats (300 to 350 g) purchased from Charles River Laboratories . The experimental protocol was approved by the Animal Ethics Board at the University of Saskatchewan conformed to the Guide for the Care and Use of Laboratory Animals stipulated by the Canadian Council on Animal Care and the National Institutes of Health publication.® Injection i.v.) was obtained from Novartis Pharmaceuticals, Canada Inc. , while tacrolimus  was purchased from Astella Pharma Canada Inc. . Isoflurane and heparin sodium were obtained from Abbott Laboratories Limited and Sandoz, Canada Inc., respectively .Acetylcholine chloride, phenylephrine hydrochloride, sodium nitroprusside, and all the salts used in the preparation of Krebs buffer were of analytical grade obtained from Sigma-Aldrich Canada Ltd. . Cyclosporine A  maintained under a resting preload tension of 2 gr. as described previously [80 level) of α1 selective agonist, PE (1 μM). Once the steady state tonic response was attained, the tissues were washed in normal Krebs buffer for a period of 1 hr, and the response to the same concentration of PE was repeated to ascertain that the sustained tonic response to PE is reproduced. Then, a fixed concentration of ACh (10 μM) was added to assess the extent of vasodilatation in PE-constricted rings. If vasodilatation to ACh was >90%, it was considered as an endothelium-intact preparation since ACh evokes vasodilatation only in vessels with intact endothelium. Once a steady tonic response was reached following the addition of PE, cumulatively increasing concentrations of either ACh (1 nM–10 μM) or SNP (1 nM–10 μM) were added in such a way that the next concentration was added only after the response to the previous concentration had plateaued. The data collected were considered as control (before treatment) and compared with data obtained after treatment of drugs in the same tissue since PE-evoked responses could be reproduced in the same preparation as long as the inhibitory drug is exposed only once. Tissues were washed 3 times and allowed to recover by repeated washing for a minimum of 1 hr. The same tissues were incubated with either CsA (1 mg/mL) or Tac (0.1 mg/mL) in Krebs buffer for 30 min. Then, the tissues were constricted with PE (1 μM) to reach steady state tonic response, and cumulatively increasing concentrations of either ACh or SNP were repeated in the presence of either CsA or Tac until the maximal inhibition (Imax) of steady state tonic vasoconstrictor response to PE could be reached. Thus, the changes in isometric tension evoked by ACh and SNP in the presence or absence of both CsA and Tac in PE-constricted rings were determined in the vessels of the same rat (n = 6). The tension responses were recorded in gram on a chart programme (Chart V5.0.1) using a Powerlab/8SP data acquisition system .eviously . Adequat50) as well as the percentage of maximal inhibition (Imax) values for each condition of incubation could be generated from each concentration-inhibition response curve (CRC) using Prism software . The data obtained using vessels isolated from one rat for a specific condition of incubation were pooled, and the mean value from that rat was considered as n = 1. Then, similar experiments were replicated with blood vessels isolated from 6 rats (n = 6). Thus, the final mean ± SEM values shown in the results section represent the data gathered from several rats. The differences in mean ± SEM values between different conditions of incubation were analysed using one-way ANOVA, followed by Tukey post hoc test, and the data were considered significant when the P value was <0.05. However, for assigning the level of significance, the closest P value reached was provided in The inhibition of tension evoked following the additions of each concentration of ACh or SNP in PE constricted vessels were normalized as the percentage inhibition of steady state tonic response evoked by PE. Since the inhibitory effect reached closer to 100%, the concentration of either ACh or SNP required to produce 50% of the maximal inhibition (ICAddition of either the vehicle (peanut oil 10%) in which higher concentrations of both CsA or Tac were prepared or the addition of appropriate concentrations of either CsA (1.0 mg/mL) or Tac (0.1 mg/mL) maintained in the organ bath for a period of 30 min in Krebs buffer failed to affect the basal tone, while PE-evoked steady tonic responses of TA or AA portions of rat aortic rings were enhanced and comparable (data not shown).μM in both TA and AA. The CRC determined from several experiments is shown  than the control value for ACh (98% ± 1%). The acute inhibitory effect of CsA was so profound in endothelium-intact TA rings, and this was similar to the abolition of the inhibitory effect of ACh seen in endothelium-denuded vessels , Tac incubation failed to affect the maximal inhibitory effect of ACh as the Imax value was not different between control versus Tac treatment groups . The Imax value was also reduced from complete inhibition (100% ± 2%) to a level of 58% ± 5%, and the inhibitory effect was significant  with right-ward shift in the CRC to ACh , and the Imax was reduced from 100% ± 2% to 80% ± 6%  incidence of hypertension in patients receiving Tac treatment [P < 0.05) than CsA when augmentation of arterial stiffening and small and large artery compliance index were considered following CsA and Tac treatment in renal transplant recipients receiving these two agents [in vitro study is discussed in the light of the above clinical observations. The major finding reported in the present study is the observation that CsA inhibits endothelium-dependent vasodilatation to a much greater extent than Tac, and this effect is more pronounced in thoracic than in abdominal region of the rat aorta. The acute reatment –16. Anoto agents . The sigin vitro studies using isolated VSM preparations. The possible mechanism by which the CNIs such as CsA and Tac could alter the phosphorylation states of serine-threonine residues from the endothelial No synthase (eNOS) leading to reduced expression and activity of eNOS. Thus, decrease in NO generation in the endothelial cells has been documented previously [Both CsA and Tac are well-established CNIs that act as immunosuppressive agents by inhibiting calcineurin, a calcium, and calmodulin-dependent serine-threonine protein phosphatase 3 in T lymphocytes . Elevatieviously , 8. Simieviously , 11. Howin vitro effects of CsA (1 mg/mL) and Tac (0.1 mg/mL) is appropriate and justified. We demonstrate that addition of such optimal low concentration of CsA for 30 min led to abolition of endothelium-dependent vasodilator responses to ACh in thoracic rat aortic rings whereas incubation with Tac for the same duration led to only partial inhibition of the responses. Moreover, Tac was much less potent as an inhibitor of ACh responses in the abdominal portion of the aortic ring. A regional difference in vasculotoxic effect of CsA with profound inhibition of endothelium-dependent (ACh-mediated) and marginal endothelium-independent (SNP-induced) vasodilatation in thoracic but not in abdominal region of aortic rings has been previously demonstrated [in vitro examination of tension responses following chronic oral administration of CsA to normotensive Sprague-Dawley rats at a dose of either 20 mg/kg for a prolonged period of either ten days or administration of a dose of 50 mg/kg for 7 days. After this treatment, the tissues were harvested, and in vitro responses were determined. In contrast, in our in vitro study we harvested both TA and AA from normal SD rats and mounted in organ bath; later we have taken vascular dilator response before and after exposure of CNIs for 30 min with TA and AA rings. Our data show that the inhibitory effect of CsA was more profound in TA as compared to AA. Verbeke et al. could not provide a convincing explanation as to why CsA selectively attenuated the endothelium-dependent ACh mediated vasodilatation in TA but not in AA of rat [α1 adrenoceptor agonist) in either TA or AA; however, response pattern was similar between TA and AA. Others have also shown that there are no segmental differences in vasoconstrictor responses evoked by PE between thoracic and abdominal rat aortic rings since PE activates one subtype of α1D adrenergic receptors in either regions [in vitro effects of CNIs are linked to inhibition of endothelium-dependent vasodilatation especially after incubation for 30 min in organ bath.It is now well established that Tac is 10 to 100 times more potent than CsA as a CNI . Thus, tnstrated . In thisA of rat . Moreove regions . Thus, o2) to maintain vasodilatation even if the NO release is blunted [2+ dependent EDHF activation that is linked to small and intermediate conductance Ca2+activated K+channels (SKCa and IKCa) in the descending portion of the rat aorta. NO mediates the vasodilator response to ACh in TA. CsA and Tac inhibit NO-mediated vasodilatation to ACh due to serine-threonine phosphorylation of eNOS. It is likely that the effect of CsA is profound in reducing eNOS activity while Tac is relatively less sensitive in the thoracic region following its acute CNI incubation. This is consistent with previous finding that CsA affect vascular function [in vitro incubation of Tac with mouse aorta in organ bath showed impaired endothelial-dependent relaxation response to ACh which was linked to altered intracellular calcium release affecting eNOS [It is now established that ACh-induced endothelium-dependent vasodilatation is mediated solely by the release of NO in major conduit vessels such as aorta , 23. As  blunted . Thus, i blunted –28. CNIsfunction , 30. Thefunction , 31. Preing eNOS . Clearlyin vitro functional study reveals that CsA is more potent than Tac in the inhibition of endothelium-dependent, ACh-induced vasodilatation in both TA and AA. There are regional variations in the responses to CNIs with TA being more sensitive to blockade than AA. In addition, we also found that CNIs did not affect the SNP-induced vasodilatation in thoracic and abdominal aorta.Our short term