Studies in murine models and human populations have indicated that the collagen-rich granulomatous response against parasite eggs trapped in the liver is associated with the development of severe hepatosplenic schistosomiasis, characterized by periportal fibrosis and portal hypertension. The role of the humoral response in parasite susceptibility has been well established, but its participation in disease severity remains poorly understood. In this work, we evaluated the relationship between parasite-reactive IgE and IgG levels and schistosomiasis morbidity in infected patients with similar parasite burdens.Schistosoma mansoni-infected individuals were subjected to clinical examination and abdominal ultrasound analysis. IgG reactivity and IgE concentration against Schistosoma mansoni soluble egg antigens (SEA) and adult worm antigen preparation (SWAP) were evaluated by ELISA assay. Multivariable linear regression models were used to evaluate the relationship between parasite-reactive antibodies and the co-variables investigated. The study population showed low parasite burden (median 30 eggs/g feces), constant re-infection, and signs of fibrosis was detected in more than 30% of individuals. Most infected individuals showed IgG reactivity, and the median concentrations of IgE anti-SEA and anti-SWAP antibodies were 1,870 and 1,375 ng/mL, respectively. There was no association between parasite burden and antibody response or any parameter of disease severity. However, IgG anti-SWAP level was positively associated with morbidity parameters, such as spleen size and thickness of portal vein at the entrance and secondary branch. In contrast, the data also revealed independent inverse correlations between concentration of parasite-reactive IgE and gallbladder wall thickness, a marker of fibrosis in schistosomiasis.Ninety-seven The data indicate that IgG anti-SWAP is positively associated with severe schistosomiasis, independently of parasite burden, while high production of parasite-specific IgE is associated with mild disease in the human population. Antibody profiles are good correlates for schistosomiasis severity and could be tested as biomarkers of disease severity. Schistosoma mansoni is the most prevalent species of the Schistosoma genus infecting human beings. Infection with this organism causes intestinal and hepatic schistosomiasis in more than 100 million individuals that primarily live in sub-Saharan Africa, the Caribbean and South American areas, including Brazil Schistosomiasis mansoni, most infected individuals are asymptomatic or have mild clinical manifestations. However, in a minority of infected individuals, infection with this parasite can lead to severe hepatosplenic schistosomiasis, characterized by periportal fibrosis, portal hypertension, gastrointestinal bleeding and death + T cells, leading to tissue eosinophilia and the activation of alternatively activated macrophages and myofibroblasts, which can increase extracellular matrix production and collagen deposition; this inflammation may also cause extensive portal fibrosis and obstructive vessel lesions and increase portal pressure Most of the morbidity related to chronic schistosomiasis is associated with hepatic and intestinal granulomatous inflammation induced by the parasite eggs that become trapped in these tissues. Granulomatous inflammation is dependent on CD4Schistosoma egg deposition induces a type-2 immune response, which is characterized by the production of IL-4, IL-5 and IL-13 cytokines that, in addition to IL-10, has been associated with the down-modulation of the initial type-1 immune response and granuloma formation S. mansoni infected patients presenting with severe fibrosis have elevated levels of the chemokine CCL3 S. japonicum infected patients from the Philippines In the murine model, Schistosoma infection on the progression of clinical disease has been poorly investigated. The importance of B cell and antibody responses in the pathology associated with schistosomiasis has been suggested from experimental infections of S. mansoni in B cell-deficient mice S. mansoni-infected patients selected from the endemic area of Corrego do Choro, Padre Paraíso city, Minas Gerais.In contrast to the amount of knowledge about the role of cytokines in granuloma formation and their association with disease severity, the participation of antibody responses against S. mansoni infection received specific treatment , and other diagnosed diseases were treated or directed for specialized treatment.The present study was reviewed and approved by the Ethical Committee of Federal University of Minas Gerais, Brazil (number 274/05). At the time of data collection, all participants or their legal guardians were required to sign an informed consent form. Independently of participation in the study, patients with confirmed S. mansoni antigens used in the experiments mice were experimentally infected as detailed in antigen preparation. All animal procedures were approved by the animal-care ethics committee of the Federal University of Minas Gerais (Protocol # 158/2008) and were performed under the guidelines from COBEA  and strictly followed the Brazilian law for “Procedures for the Scientific Use of Animals” (11.794/2008).To obtain S. mansoni-infected residents of Jequitinhonha Valley, in the northeast of Minas Gerais state (Brazil), an area endemic for schistosomiasis. The initial study evaluated 741 inhabitants 5 years of age or older from rural communities of Jequitinhonha Valley and revealed a prevalence of schistosomiasis of 73% Leishmania infection The subjects used in this study were selected among S. mansoni infection was determined based on egg counting of two thick stool smears using the Kato-Katz technique The methodology employed for the data collection has previously been described in detail elsewhere S. mansoni-infected patients that were 14 to 68 years old and had provided plasma samples that were kept at −70°C, totalizing 97 subjects. Plasma samples from 8 non-infected donors were also used for IgG reactivity controls. For the individuals selected for the current study, morbidity was evaluated by clinical aspects and by the quantitative method defined by Niamey’s protocol  that had been maintained in the Laboratory of Schistosomiasis (ICB/UFMG) by successive passages in Biomphalaria glabrata and hamsters (Mesocricetus auratus).To obtain adult worms and eggs for antigen preparation, Swiss mice were infected subcutaneously with 100 cercariae of S. mansoni worms  were recovered by perfusion of the circulatory system of 6-week-infected mice Adult Schistosoma mansoni eggs were recovered from the livers of 6-week-infected mice, and the clean egg solution was ground in cold PBS to obtain the soluble egg antigen (SEA) S. mansoni SEA or SWAP as described by March et al. S. mansoni-infected patients. Briefly, the Sepharose was extensively washed with 1 mM HCl solution, followed by 3 washes with conjugation buffer . Each antigen preparation (SEA and SWAP) was dialyzed overnight against conjugation buffer and added to the washed Sepharose at 5 mg antigen/mL Sepharose. The Sepharose/antigen mixture was incubated for 4 h at room temperature, followed by 18 h at 4°C under constant and slow agitation. After protein conjugation, the Sepharose was washed with conjugation buffer and blocked with 1 M ethanolamine (pH 8.0) for 2 h. Next, the antigen-conjugated Sepharose was submitted to 6 wash cycles alternately using an acidic buffer  and basic buffer  and then stored in a borate buffer  containing 20% ethanol to prevent microbial growth. As a control for non-specific binding of plasma protein, we also prepared Sepharose columns with no conjugated antigens that were washed and blocked as above.Sepharose columns  were conjugated with Two hundred µL each of SEA-Sepharose, SWAP-Sepharose or control-Sepharose were transferred to 1.5 mL Eppendorf tubes. Plasma samples from each patient were diluted 1∶100 in PBS buffer, and 400 µL of the diluted plasma was added to the Eppendorf tube containing SEA-Sepharose, another 400 µL of the diluted plasma was added to the SWAP-Sepharose tube and 400 µL of the diluted plasma was added to the control-Sepharose tube. Tubes containing Sepharose with plasma were vortexed for 1 min and kept at 4°C for 24 h with occasional agitation. Each tube was then centrifuged , and the supernatant was gently collected for quantification of total IgE and reactive IgG. At the end of the adsorption process, we obtained a sample of SEA-adsorbed plasma, a sample of SWAP-adsorbed plasma and a sample of control-adsorbed plasma from each patient.S. mansoni antigens in the plasma of each patient was determined by enzyme-linked immunosorbent assay (ELISA). Briefly, 96-well plates  were coated with 100 µL/well of 0.1 M carbonate-bicarbonate buffer (pH 9.5) containing 10 µg of antigen/mL (SEA or SWAP) and incubated overnight at 4°C. The plates were blocked for 1 h with PBS buffer containing 1% bovine serum albumin . After the blocking procedure and between each incubation step, the plates were washed 5 times with PBS containing 0.05% Tween (Sigma). One hundred microliters per well of diluted plasma from each patient was added to the plate and incubated for 1 h at room temperature. SWAP- and SEA-adsorbed plasma samples, as well as the control-adsorbed plasma samples, from each patient were tested. For the ELISA test, plasma samples collected after the adsorption procedure were diluted 1∶2 in PBS containing 1% BSA. To establish the reactivity threshold in plasma of infected patients, plasma samples were collected from 8 health volunteers with no report of previous infection for schistosomiasis, and the absorbance average plus 2 standard deviations from these samples were used as a reference. Bound IgG was detected by horseradish peroxidase-conjugated goat anti-human IgG (Sigma) diluted 1∶5,000 in PBS, followed by the addition of substrate solution  containing hydrogen peroxide in 0.05 M phosphate-citrate buffer (pH 5.0). The reaction was allowed to proceed for 15 min at room temperature and stopped with 4 N H2SO4 (50 µl/well). The absorbance was measured at 492 nm using an automated ELISA reader . Each plasma sample was tested in duplicate. To evaluate the reproducibility of this assay, 15% of the plasma samples were randomly selected to be retested; each duplicate received a different number from the original sample.The presence of IgG reactive against S. mansoni-adsorbed plasma (SEA and SWAP) and control-adsorbed plasma from each patient was diluted 1∶2 in diluent solution , and 100 µL/well was added to the plates. Known concentrations of purified human IgE  were added to each plate to obtain a standard curve. Plasma samples and standards were incubated for 1 h at room temperature. Immunoglobulin E bound to the plates was detected by the addition of peroxidase-conjugated anti-human IgE  at 1∶40,000 dilution in a diluent solution, followed by the addition of substrate solution . After 30 min, the reaction was stopped with 100 µL of 2 N sulfuric acid solution, and absorbance was determined using a 492 nm filter in the ELISA reader . For each patient, the amount of total IgE in control-adsorbed plasma, as well as in SEA- and SWAP-adsorbed plasma, was quantified in duplicate. The difference between the amount of total IgE obtained in the control- and SEA-adsorbed plasma from the same patient was considered to be the amount of SEA-specific IgE present in the patient. Similarly, the difference between the control- and SWAP-adsorbed plasma represented the amount of SWAP-specific IgE To quantify the total IgE concentration in the plasma of the study population, a commercial kit  with an established protocol from the manufacturer optimized for the study conditions was used. Briefly, 96-well plates (Nunc-Maxisorb) were sensitized with 1 µg/well of capture antibody for IgE  in carbonate-bicarbonate buffer (pH 9.6) for 1 h at 4°C and blocked for 45 min with 200 µL/well block solution (50 mM Tris-HCl buffer containing 0.14 M sodium chloride and 1% BSA). Between each step, the plates were washed 3 times with Tris/NaCl/Tween wash buffer (50 mM Tris-HCl buffer containing 0.14 M sodium chloride and 0.05% Tween 20). 2 test, means were compared using Student’s t-test or analysis of variance (ANOVA), and the Kruskal Wallis test was used to compare medians. Additionally, Spearman correlation analysis was performed for continuous variables. Linear regression analysis were used to evaluate the relationship between each immunoglobulin (IgE and IgG) reactive to egg (SEA) and adult worm (SWAP) antigens of S. mansoni and the co-variables collected . IgE values were transformed into base 10 logarithms.Databases were generated using EPI-INFO version 6.04, and statistical analyses were performed using STATA version 11.0 software . Normality tests were determined with the Shapiro-Wilk test, and non-parametric variables were normalized. Categorical variables were compared using the χInitially, simple linear regression analysis was used to compare each immunoglobulin with all of the co-variables collected. Variables with p values <0.25 were selected to construct the multivariable linear regressions models and US-measurements were used only as continuous values for the final model. Moreover, variables with low frequency and that showed co-linearity were excluded from multivariable analysis. Full models for each immunoglobulin were constructed with all of the independent variables selected for univariate analysis, and modeling was carried out through a backward process. The only variables remaining in the model were those that were statistically significant with a confidence interval (CI) of 95% and p-value <0.05. Results are reported as linear regression parameter estimates, along with 95% CIs for the effect of independent variables for the immunoglobulin of interest. Homoscedasticity of the final models were evaluated.Schistosoma treatment. Seventy-seven individuals (79%) from the study population showed less than 100 Schistosoma eggs eliminated per g of feces (low parasite burden), with a mean of 81.1±163.4 and median of 30 eggs/g feces. Among the 97 individuals evaluated in the study, there was no association between parasite burden and age, sex or gender . The ages ranged from 14–68 years, and age was equally distributed throughout the groups; the mean age was 33.3 years (SD 5.8), and the median was 30 years (IQR 26). The evaluated individuals weighed on average 52.8 kg (SD 10) and had a mean height of 157 cm (SD 9.8). At the time of sample collection, the population had no access to treated water or sewage treatment, and 32 individuals (33%) reported previous r gender .The clinical examination revealed that among the 97 individuals evaluated, 7 (7.2%) reported previous hematemesis, and 2 had collateral veins detected during clinical examination. The spleen of 9 patients was enlarged, and 4 of them also showed hard consistency. Moreover, 50 individuals (51.6%) showed enlarged livers, with 10 of them having a hard consistency and 4 individuals showing gross irregularities (pseudo-nodules) of the liver surface.Schistosoma-related morbidity, only 5 individuals showed spleen sizes larger than 120 mm, and 4 had right liver lobes that were reduced in size. However, at least 30% of the study population showed some signal of fibrosis, such as thickening of the portal vein and gallbladder wall  showed a significant decrease in IgE response  was observed . Individresponse .To investigate whether the antibody response was related to schistosomiasis morbidity, the concentration of parasite-reactive IgE or the level of IgG reactivity was associated with clinical and ultrasound parameters of disease. The level of IgG anti-SWAP was significantly higher in patients with livers of hard consistencies and gross irregularities on the liver surface. Moreover, increases in the size of the left lobe of the liver, as defined by clinical examination, were positively correlated with the level of IgG anti-SEA but not with IgG anti-SWAP, while individuals that had palpable spleens during clinical examinations showed higher levels of IgG anti-SWAP (data not shown). In contrast, there was no association between the concentration of IgE reactive against SEA or SWAP with any of the clinical parameters evaluated.Liver size as determined by ultrasound examination showed no association with the level of IgG anti-SWAP or anti-SEA, but there was a positive association between the level of IgG anti-SWAP and the size of the spleen . There wS. mansoni infection was evaluated by ultrasound by measuring the portal vein diameter and thickness at its entrance into the porta hepatis and its bifurcation inside the liver, as well as the thickness gallbladder wall. The association between these disease parameters and antibody production was also estimated. A positive association was observed between IgG anti-SWAP and the diameter and thickness of the portal vein at the entrance and its bifurcation  Among the infected individuals, there was no association between parasite burden and antibody response or disease severity. iii) There was a positive association between parasite-reactive IgG (mainly anti-SWAP) level and both spleen size and portal vein thickness. iv) The data also revealed inverse correlations between the concentration of parasite-reactive IgE and gallbladder wall thickness, an important marker of fibrosis in schistosomiasis.This cross-sectional study evaluated the possible association between antibody production, IgG and IgE, and schistosomiasis in a naturally infected population. The major findings of the present study can be summarized as follows: i) A human population exposed to low In the present work, we quantified for the first time in a human population the concentration of IgE against schistosome antigens without interference from other isotypes, such as IgG4. The procedure is essential to evaluate the real role of IgE in the infection, since B cells can switch sequentially from an IgG4-producing B cell into an IgE-producing B cell. Therefore, high IgG4 production, which may be more than 100-fold higher than IgE antibodies, can interfere with the detection of parasite-specific IgE Schistosoma infection have shown an inverse correlation between host age and parasite burden S. mansoni hyperendemic areas, Bina and Prata S. mansoni with low parasite burdens and no association between parasite burden and disease severity, such as the area evaluated in the present study, offer a unique opportunity to evaluate the effect of the immune response on disease severity independently of infection intensity. More importantly, even with low parasite burdens, more than 30% of the individuals evaluated in this study had some measureable level of fibrosis, as shown in The individuals evaluated in this study had a homogeneous gender and age group distribution, and no association between gender, age group and parasite burden was observed. Although most population analyses performed in high endemic areas for Schistosoma-infected populations have reported that anti-Schistosoma IgG4 levels in infected children are associated with higher parasite burdens and parasite susceptibility Regardless of the antibody response to this disease, most of the studies on S. mansoni+ B cells with resistance to S. mansoni can be detected even in children Schistosoma treatment had significantly lower levels of IgE, independently of age, sex or parasite burden. These data are suggestive of a protective role for IgE in the study population.In contrast to IgG, data from endemic areas of schistosomiasis have shown that the reactivity of anti-SWAP IgE increases with age S. mansoni-endemic area of Bahia, Brazil, also showed no significant difference in the levels of Schistosoma-specific IgE between individuals with different parasite burdens, but the authors found that levels of anti-adult worm IgG4 and IgE/IgG4 ratios were inversely associated with S. mansoni parasite burden S. haematobium-endemic area Schistosoma infection could be related to the IgE/IgG4 balance rather than the level of production of a single isotype.Recent data from a large casuistic study in an Schistosoma infection, there have been very few studies showing the relationship between specific antibody production and parameters of disease severity. Using ultrasound measurements to categorize patients infected with S. mansoni, Tawfeek et al. S. mansoni-endemic areas also showed that levels of anti-SEA IgG4 were significantly higher in sera from patients with fibrosis as detected by ultrasonography compared with other patients S. mansoni antigens, SEA and SWAP, is also positively associated with severe forms of schistosomiasis, defined by clinical examination and by ultrasound measurement. Specifically, we identified an independent positive association between levels of anti-SWAP IgG and portal hypertension and fibrosis markers, such as thickness of the portal vein at its entrance into the porta hepatis and its bifurcation inside the liver and spleen size measured by ultrasound. Unsurprisingly, parasite-reactive IgG was also associated with disease because IgG4 recognizing SEA and SWAP antigens represent the most prevalent IgG isotype in Schistosoma-infected patients Although there have been many epidemiological studies correlating antibody production with host susceptibility/resistance during The association of IgG4 with severe forms of schistosomiasis has been justified by the increased susceptibility of the host to infection with the parasite; this finding indicates that an excess of IgG4 would block the protective effect of IgE, favoring parasite establishment and increasing egg deposition into host tissue and the progression to severe forms of the disease S. mansoni reinfection, associating IL-10 production with reinfection susceptibility. Interestingly, IL-10 is presumably needed to drive the differentiation of IgG4-switched B cells to IgG4-secreting plasma cells Schistosoma pathology, with IL-10 acting as an anti-inflammatory factor during granuloma formation in the liver Recent experimental work in mice Schistosoma-induced liver pathology was previously demonstrated by exacerbated hepatic granuloma formation and fibrosis detected in B cell-deficient mice, mice lacking the common FcRγ chain and mice deficient in FcεRI, the high-affinity receptor for IgE + cells, such as FcR-bearing macrophages, DCs, B cells, basophils, mast cells and neutrophils. IC-FcγR activation could inhibit granuloma formation by generating the production of immunoinhibitory molecules, such as IL-10, prostaglandins and histamine, secreted by one or more of these types of cells S. mansoni infection, alternatively activated macrophages in the liver bind to immune complexes via FcRs and assume regulatory/anti-inflammatory roles. In the human population, a possible role for immune-complexes in granuloma formation/modulation was also suggested by experimental work that demonstrated that circulating immune complexes purified from the serum of patients with mild clinical forms of schistosomiasis downregulate the reactivity of peripheral blood mononuclear cells to Sepharose-conjugated parasite antigens, resulting in diminished granuloma reaction S. mansoni pathology was not reported for humans. Interestingly, histamine, which is normally associated with the induction of inflammation, has been shown in previous studies to downregulate schistosome-mediated granuloma formation Our data also showed, for the first time, an independent inverse correlation between anti-SEA and anti-SWAP IgE and the wall thickness of gallbladder, an important marker of fibrosis Schistosoma-induced fibrosis. These data indicate an important role for antibodies in granuloma modulation, and consequently correlating with schistosomiasis severity.In summary, the current work demonstrated that parasite-reactive IgG levels are associated with signals of disease severity independently of parasite burden and that IgE concentration is inversely associated with Checklist S1STROBE Statement–Checklist of items included in reports of cross-sectional studies.(DOCX)Click here for additional data file.


The relationships between total serum IgE levels and gene expression patterns in peripheral blood CD4+ T cells  are not known.Peripheral blood CD4+ T cells from 223 participants from the Childhood Asthma Management Program (CAMP) with simultaneous measurement of IgE. Total RNA was isolated, and expression profiles were generated with Illumina HumanRef8 v2 BeadChip arrays. Modeling of the relationship between genome-wide gene transcript levels and IgE levels was performed in all subjects, and stratified by sex.2) in IgE measurement . Sex stratified analyses revealed that the correlation between IL17RB and IgE was restricted to males only . Significant correlation between gene transcript abundance and IgE level was not found in females. Additionally we demonstrated substantial sex-specific differences in IgE when considering multi-gene models, and in canonical pathway analyses of IgE level.Among all subjects, significant evidence for association between gene transcript abundance and IgE was identified for a single gene, the interleukin 17 receptor B (IL17RB), explaining 12% of the variance (rOur results indicate that IL17RB may be the only gene expressed in CD4+ T cells whose transcript measurement is correlated with the variation in IgE level in asthmatics. These results provide further evidence sex may play a role in the genomic regulation of IgE. Total serum immunoglobulin E (IgE) is a risk factor for both the development of  and diseSex is a critical determinant of IgE level, with males having a stronger tendency towards higher total IgE than females . Sex-relBased on these findings we hypothesized that genome-wide CD4+ T cell microarray analyses would provide unique insights into genes that correlate with the variability in IgE level, and that sex would be an important modifier of these correlations. To test these hypotheses we explored the relationship between total serum IgE levels and genome-wide gene expression transcript abundance in a cohort of 223 young adults with asthma, both in all subjects and within each sex specifically.6 total cells. Pilot studies confirmed CD4+ cell yields of ~4 × 106 at ≥ 95% purity per collection. Total RNA was extracted using the RNeasy Mini Protocol   5-17) of the number of transcripts that should overlap by chance alone. At a more liberal threshold for inclusion of the results from females (p < 0.1), only 38 of 1205 unique transcripts (3.1%) overlap. At a more conservative threshold (p < 0.01) there were no transcripts common to both males and females.2 of 0.09 in the 89 CAMP females).We considered whether our findings of association in males but not females could be attributable to differences in sample size. Though power was somewhat lower in females due to the smaller number of subjects , power was very high (>91%) to detect correlations half as strong as those observed among males , hexose-6-phosphate dehydrogenase (H6PD), IL17RB, poly(A) binding protein interacting protein 1 (PAIP1), and PVALB  could both confirm our findings of association from single gene regressive strategies, and could identify additional determinants of total serum IgE levels. Among all subjects, the optimal multivariate model included 17 transcripts (including IL17RB), of which 16 map to unique HUGO gene identifiers . Notably, there were no significant canonical pathways in common between males and females. Additional information about the canonical pathway analyses, including lists of differentially expressed genes within the pathways are included in Additional Files To better understand the combinatorial effect of multiple gene transcripts on IgE regulation we performed canonical pathway analyses. Of the 763 transcripts demonstrating nominally significant (p < 0.05) correlation with total serum IgE, 652 85%) map to a unique HUGO gene id and were used for IPA canonical pathway analysis. Among all subjects, enrichment for six pathways was observed (Fisher's exact p < 0.05) including the antigen presentation pathway, propanoate metabolism, interferon signaling, primary immunodeficiency signaling, the coagulation system, and the pantothenate and CoA biosynthesis pathway  identified in a recent genome-wide association study of IgE [This is the first study to correlate genome-wide CD4+ T cell transcript abundance with IgE level in humans and is, to our knowledge, the largest gene-expression study of as asthma-related quantitative phenotype published to date. Our results indicate that IL17RB may be the only gene expressed in CD4+ T cells whose transcript measurement is correlated with the variation in IgE level in asthmatics. Given that IgE level is strongly influenced by environmental factors  it is surprising that 12% of the variation in IgE can be explained by IL17RB transcript measurement, a correlation that is substantially greater than that explained genetic determinants from three significant genomic regions  but has much higher binding avidity to interleukin 17 E  . While s2 = 19%), but not in females (r2 = 3%); evidence for a multivariate gene signature of total IgE levels in males, but not in females; and discrete, non-overlapping canonical gene pathway enrichment in each sex. Notably, we found limited statistical evidence to suggest that there is significant overlap between CD4+ T cell genes whose transcript measurements correlate with IgE level in both male and female asthmatics. Our work stands with a body of literature now implicating an important role for sex in the heritability, genome-wide linkage analyses, [this report) of IgE, and strongly support the notion that genetic and genomic studies of IgE should consider the role of sexual dimorphism.Another finding of our analyses is that the magnitude of the relationship between patterns of CD4+ lymphocyte gene expression and total serum IgE levels is substantially different between males and females. Several lines of evidence are provided, including: strong association of IL17RB transcript abundance with IgE levels in males  to detect the male-specific associations in our female sample, replication in larger sample sizes could help to determine if the lack of overlap we note between males and females is primarily an issue of the magnitude of the correlations or of true differences in the genomic regulation of IgE. In addition, our gene expression analyses include gene transcript measurements from unstimulated peripheral blood CD4+ lymphocytes in a population of adolescents and young adults recruited on the basis of childhood asthma. We urge caution in extrapolating our findings to other populations and cell types. Finally, while an assessment of IL17RB protein levels would provide additional supportive information, we do not have intact CD4+ lymphocytes enabling suitable cell surface protein quantification.TSLP polymorphisms on the regulation of specific, and total serum IgE in children with asthma [While the precise role that sex plays in the genomic regulation of IgE is unknown, sex differences in the immune environments experienced by CD4+ T cells are well described. In animals, females are more likely to develop a Th1 response after antigen challenge, except during pregnancy when a Th2 environment dominates . Although asthma . This ish asthma .-6), particularly in light of prior studies suggesting the importance of interferon-γ (INFG) in the female  allergic response [Though we have limited evidence that single genes, or genetic models strongly correlate with IgE level among females, it is intriguing that the canonical pathway most significantly correlated with IgE in females was the interferon signaling pathway  correlated with total serum IgE in all subjects.Click here for fileCanonical pathways (and genes) correlated with total serum IgE in males.Click here for fileCanonical pathways (and genes) correlated with total serum IgE in females.Click here for file


Activities of transfected transporters were distinguished from those of endogenous ones by working at 37°C. fNKCC1 and fNKCC2A activities were highest after pre-incubation of cells in hypotonic low-[Cl−] media to reduce cell [Cl−] and volume during flux measurement. Phosphorylation of both transporters more than doubled. Pre-incubation with ouabain also strongly stimulated fNKCC1 and fNKCC2A and substantially increased phosphorylation, whereas pre-incubation in Na+-free media maximally stimulated fNKCC1 and doubled its phosphorylation, but inhibited fNKCC2A, with a small increase in its phosphorylation. Kinase inhibitors halved phosphorylation and activity of both transporters whereas inhibition of phosphatases with calyculin A strongly increased phosphorylation of both transporters but only slightly stimulated fNKCC1 and inhibited fNCCC2A. Thus kinase inhibition reduced phosphorylation and transport, and transport stimulation was only seen when phosphorylation increased, but transport did not always increase with phosphorylation. This suggests phosphorylation of the N-termini determines the transporters' potential capacity to move ions, but final activity also depends on other factors. Transport cannot be reliably inferred solely using phospho-specific antibodies on whole-cell lysates.Na-K-2Cl cotransporters help determine cell composition and volume. NKCC1 is widely distributed whilst NKCC2 is only found in the kidney where it plays a vital role reabsorbing 20% of filtered NaCl. NKCC2 regulation is poorly understood because of its restricted distribution and difficulties with its expression in mammalian cell cultures. Here we compare phosphorylation of the N-termini of the cotransporters, measured with phospho-specific antibodies, with bumetanide-sensitive transport of K Both are potently and selectively inhibited by the loop-diuretic bumetanide which can be used to identify and characterise transport. NKCC1 is found mainly in the basolateral membranes of secretory epithelia where it facilitates the entry of Na+ and Cl− into cells from interstitial fluid. NKCC1 is also widely expressed in non-epithelial cells where it helps regulate cell composition and volume. NKCC2 is found specifically in the apical membranes and subcellular vesicles of cells in the thick ascending limb of Henle's loop (TAL) in the kidney. Here, as three splice variants , it reabsorbs about 20% of filtered NaCl from the urine, with NKCC2A distributed throughout the whole TAL. The regulation of NKCC1 has been extensively studied. Under conditions where transport is stimulated  the cotransporter becomes phosphorylated on three threonine residues ) in a key regulatory domain in the N-terminus of the transporter Na+ pump.In this paper we focus on the relationship between phosphorylation and transport rate and the effects of ouabain on transport. Given the problems of working with NKCC2, the ability to infer its activity from measures of protein phosphorylation using suitable antibodies and thus obviating the need for technically demanding transport studies, would be of immense practical benefit in studying kidney function, for instance its role in essential hypertension. However, for NKCC1 there is evidence that some factors, for instance those which alter its interactions with the cytoskeleton, may influence transport rate independently of cotransporter phosphorylation Ferrets are used to model human cardiovascular physiology, for instance study of NKCC1 in ferret erythrocytes has facilitated the understanding of the transporter + orthovanadate (Sigma-Aldrich) was prepared as described elsewhere 86RbCl was from Perkin-Elmer. Incubation media pH was adjusted to 7.4 with NaOH at the appropriate temperature.Chemicals were of analytical grade, and water was of Milli-Q quality (Millipore). Drugs were prepared as 100× stock solutions. 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolopyrimidine , genistein (Sigma-Aldrich), calyculin A (Enzo Life Sciences) were dissolved in DMSO (Merck) whereas ouabain (Sigma-Aldrich) and bumetanide  were dissolved in water. Stock (200 mM) Na2, 1 MgCl2, 5 glucose, 15 Hepes, pH 7.4. Hypotonic, low [Cl−] medium: 67.5 Na+ gluconate, 2.5 K+ gluconate, 0.5 CaCl2, 0.5 MgCl2, 5 glucose, 15 Hepes, pH 7.4. Uptake medium: 135 NaCl, 1 RbCl, 1 CaCl2, 1 MgCl2, 5 glucose, 10 sorbitol, 0.1 ouabain, 15 Hepes, pH 7.4 and ∼1 µCi/ml 86Rb+. Lysis buffer: 50 NaF, 5 Na4P2O7, 5 EDTA, 1 Na+ orthovanadate, 1% Triton X-100, 1% protease inhibitor cocktail (Merck), and 20 Hepes, pH 7.4. Na+-free medium was similar to basic medium but with all Na+ replaced by N-methyl-d-glucamine (NMDG), and Hepes neutralised with Tris-base.Compositions (in mM) of media were as follows. Basic (isotonic) medium: 135 NaCl, 2.5 KCl, 1 CaClBamHI restriction site in the second half of the fNKCC1 gene , and cloned into the pGEM-T-Easy vector (Promega) to give pGEM-5′-f1 and pGEM-3′-f1, respectively. The 5′- and 3′-NKCC1 fragments were subcloned into pcDNA3.1 using a three way ligation to obtain the complete open reading frame: pGEM-5′-f1 was digested with KpnI/BamHI and pGEM-3′-f1 with BamHI/EcoRI and the resulting fragments were ligated into the KpnI/EcoRI sites of pcDNA3.1. From that, the complete fNKCC1 coding region was amplified using 5′-GCT GGC TAG CGT TTA GAA TTC AGC TTG GTA CCG CAG CG-3′ and 5′-TAG AAG GCA CAG TCG AGG-3′, inserted into the EcoRI site of pCI-neo (Promega) and the obtained pCI-fNKCC1 construct sequenced .Total RNA preparation from ferret kidneys and subsequent cloning of the 5′- and 3′- regions of the fNKCC1 gene were performed as described earlier Sequence data for fNKCC1 was deposited at GenBank under accession number GQ338078.2. To create a cell line stably expressing fNKCC1, HEK-293 cells were transfected with pCI-fNKCC1 using ExGen 500  according to the manufacturer's instructions and stable transfectants were selected by growth in the presence of 0.3 mg/ml Geneticin. HEK-293 cells stably expressing fNKCC2A have been described before HEK-293 cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 4 mM L-glutamine and, in case of stably transfected cells, 0.15 mg/ml Geneticin (Invitrogen). All cells were maintained at 37°C in a water-saturated atmosphere containing 95% air and 5% CO86Rb+ influx in wild-type or stably transfected HEK-293 cells grown to confluence in 96-well plates as described previously −] medium. Drugs were added to basic medium during this period. Where used, orthovanadate (1 mM) was present for the full 60 min whereas DMSO (1%), calyculin A (0.25 µM), PP1 (50 µM), and genistein (120 µM) were present for the final 30 min. In experiments on the time-dependence of the effects of ouabain or Na+-removal, ouabain (0.1 mM) or the Na+-free medium was present for the last 5–30 min. In step 2 cells were incubated for 5 min in the final pre-incubation medium (containing any drugs) or the Na+-free medium with the addition of 0.1 mM ouabain. In step 3 86Rb+ uptake was measured in uptake medium containing any drugs where indicated. Uptake was measured for 2–3 min at RT, for 1 min at 37°C, and for 7 min when bumetanide was present. In all cases the bumetanide-resistant uptake was measured using an identical protocol but with 10 µM bumetanide present during steps 2 and 3. 86Rb+ uptake was terminated by removing the uptake medium and washing cells three times in cold 110 mM MgCl2 using an automatic plate-washer . Cell 86Rb+ was determined with a Typhoon phosphor imager . At least 3 wells were used for each experimental point. Cell protein content for each construct was determined from wells which had not received 86Rb+. Cells were lysed in lysis buffer and protein concentration was determined using a BCA protein assay .NKCC activity was measured as bumetanide-sensitive + uptake assays described above, but without addition of 86Rb+. After removal of uptake medium, the plates were immediately placed on ice and cells were lysed in ice-cold lysis buffer. Lysates from four wells were combined and their protein content determined. SDS-PAGE and Western blotting were performed as described previously −] Wild-type and stably transfected HEK-293 cells were incubated at 37°C as for RbValues are given as means ± s.e.m. with n the number of independent experiments. Occasionally, it was not possible to repeat every component of an experiment because of cell instability. Data analysis under these circumstances is described in figure legends. Differences between normalised means were assessed using two-tailed t-tests, with the level for significance set at P<0.05. Non-linear regression analysis  was performed using Graph Pad Prism .80RFQV and 130RFRV) for binding SPAK and OSR1 132RVNF) for binding protein phosphatase 1 (PrP1) −] medium prior to flux measurement (in an isotonic medium) caused a large activation of transport  and a pI of 6.03. The protein has 95% and 94% similarity with human and mouse NKCC1 respectively. The threonine residues in the N-terminus regulatory domain are conserved together with the surrounding sequence. Also in the N-terminus are two consensus sites  uptake in the presence of ouabain. The latter steps are usually carried out at RT 86Rb+ uptake by fNKCC1 cells was indistinguishable from that by control HEK-293 cells, whereas it was twice as high in HEK-293 cells expressing fNKCC2A (fNKCC2A cells). Exposure to a hypotonic, low [Cl−] medium was needed to distinguish fNKCC1 from HEK-293 NKCC  and fNKCC2A (2–3 fold), and a smaller increase in that of endogenous HEK-293 NKCC as detected by phospho-specific antibodies R5 and NKCC-P , Table 110, of fNKCC1 and fNKCC2A fluxes are 2.18 and 2.15 respectively. Data in −] medium  medium  medium . This cae medium . Phospho86Rb+ uptake in cells transfected with human NKCC1 86Rb+ uptake in untransfected HEK-293 cells , whereas it increased fluxes by 33.4±4.8%  in fNKCC1 cells, but slightly reduced fluxes by 18.9±10.3%  in fNKCC2A cells. Although calyculin A increased fNKCC1 activity this represents only about 15% of the stimulation seen when cells are pre-incubated in hypotonic low [Cl−], or low [Na+] media.As regulation of NKCC activity is linked to phosphorylation of key threonine residues in the N-terminus of the cotransporter we tested the effect of different phosphatase and kinase inhibitors. Concentrations and exposure times were chosen on the basis of previous experience of these reagents on transport in other cells + orthovanadate, an inhibitor of tyrosine phosphatases (as well as a potent inhibitor of P-Class ATPases such as the Na+ and Ca2+ pumps), markedly inhibits the dephosphorylation of NKCC1 86Rb+ uptake . In contrast to these differential effects of phosphatase inhibitors, treatment of cells with kinase inhibitors, 50 µM PP1 or 120 µM genistein, reduced 86Rb+ uptake in all three cell types by approximately 50%  and a 5-fold increase in fNKCC1 phosphorylation observed at 155–175 kDa , Table 1Overall there was a poor correlation between changes in phosphorylation and transport in the presence of calyculin A. In HEK-293 cells increased phosphorylation of endogenous NKCC was not accompanied by any significant change in cotransporter fluxes, and in fNKCC1 cells, a five-fold increase in phosphorylation was accompanied by only a 30% increase in transport . For fNK−1 min−1) were observed in fNKCC1 cells following their pre-incubation in a hypotonic, low [Cl−] medium , the NKCC2 isoform which is present throughout the TAL and has similar ion affinities to NKCC1 ] medium , and sure medium , and maye medium  may resun fNKCC1 . Indeed n fNKCC1  to aboutThe links between phosphorylation of two neighbouring domains in the N-termini of fNKCC1 and fNKCC2A and transport rate (activity) were examined. Treatment of cells in a variety of ways can produce similar patterns of phosphorylation, but it is often difficult to assign these effects appropriately to those kinases or phosphatases that phosphorylate or dephosphorylate the cotransporter itself as kinase activation and phosphatase inhibition have similar effects. What clearly emerges from our studies is that inhibition of kinases reduces phosphorylation with proportional changes in transport, and that transport stimulation is only seen when phosphorylation increases from its basal level. However, it does not always follow that an increase in phosphorylation results in transport stimulation. Phosphorylation is necessary, but not sufficient. Thus phospho-specific antibodies may provide important information about cotransporter phosphorylation and the integrity of cell signalling pathways but they may not be as reliable for reporting cotransporter activity.86Rb+ uptake or cotransporter phosphorylation using kinase inhibitors Both PP1, a kinase inhibitor selectively affecting Src, Lck, RIP2, GAK, and CK1δ −] medium  by ouabain had been reported in cultured cells 2+] as the same responses were seen in cells incubated in Ca2+-free media  under these conditions, and perhaps suggesting that NKCC1 is more prone to this mode of transport than NKCC2F. It is likely that some K+-K+ exchange contributes to 86Rb+ uptake measured here. However, the ion gradients favoured net influx (rather than K+-K+ exchange) under most conditions (and cell [Cl−] was often reduced), except perhaps after long exposure to ouabain, where a rise in intracellular [Na+] might favour self-exchange. Conversely, a fall in K+-K+ exchange might explain the slight drop in 86Rb+ uptake seen in HEK-293 cells incubated in a Na+-free medium.Recently, NKCC1 and NKCC2F, expressed in + is filtered each hour, day or night, and must be reabsorbed, dietary fluctuations represent a tiny fraction of this), whereas for NKCC1 activity may have to be low at rest but be capable of rising rapidly to deal with high peak demands. Strong activation by a fall, and inhibition by a rise in cell [Cl−] or [Na+] makes physiological sense for NKCC1, but not for NKCC2 where such effects would limit the ability of the transporter to respond to changes in urine ion concentration. Here, it is important that both Cl− and Na+ are allowed to pass through the epithelium without raised cell levels unduly inhibiting further entry on the cotransporter.The differences between fNKCC2A and fNKCC1 shown in this study - ranging from fNKCC2A's higher resting activity to its reduced responsiveness to a variety of stimuli - are in line with the different locations and physiological roles of the transporters. Changes in urine ion concentrations may be the major determinant of NKCC2A activity (load-dependent) whereas there is limited scope for variation in extracellular ion concentrations for NKCC1, and the actions of hormones and cytokines may be much more important. NKCC2A has a steadier work-load (in man 1 mole of Na


The phosphorylation of virus proteins by host kinases is linked to viral replication. This leads to an inhibition of normal host-cell functions. Further elucidation of phosphorylation in virus proteins is required in order to aid in drug design and treatment. However, only a few studies have investigated substrate motifs in identifying virus phosphorylation sites. Additionally, existing bioinformatics tool do not consider potential host kinases that may initiate the phosphorylation of a virus protein.329 experimentally verified phosphorylation fragments on 111 virus proteins were collected from virPTM. These were clustered into subgroups of significantly conserved motifs using a recursively statistical method. Two-layered Support Vector Machines (SVMs) were then applied to train a predictive model for the identified substrate motifs. The SVM models were evaluated using a five-fold cross validation which yields an average accuracy of 0.86 for serine, and 0.81 for threonine. Furthermore, the proposed method is shown to perform at par with three other phosphorylation site prediction tools: PPSP, KinasePhos 2.0 and GPS 2.1.http://csb.cse.yzu.edu.tw/ViralPhos/.In this study, we propose a computational method, ViralPhos, which aims to investigate virus substrate site motifs and identify potential phosphorylation sites on virus proteins. We identified informative substrate motifs that matched with several well-studied kinase groups as potential catalytic kinases for virus protein substrates. The identified substrate motifs were further exploited to identify potential virus phosphorylation sites. The proposed method is shown to be capable of predicting virus phosphorylation sites and has been implemented as a web server  A virus is a biological agent capable of interrupting and manipulating normal functions of a cell . In humaProtein phosphorylation is a well-studied post-translational modification (PTM) process in eukaryotic cells . The prohttp://csb.cse.yzu.edu.tw/ViralPhos/.This study aims to analyze experimentally identified virus phosphorylation sites by bioinformatics analysis. We present a statistical method for identifying potential phosphorylation sites and its potential kinase substrate motifs on virus proteins. In this work, substrate motifs were identified and matched with several well-studied kinase groups as potential catalytic kinases for virus protein substrates. The identified substrate motifs were further exploited to help identify potential virus phosphorylation sites. The method is implemented as a web server, ViralPhos, accessible at Virus phosphorylation data were collected from major protein databases: virPTM , dbPTM ,12, UniPK-means clustering method based on sequence identity [K, which denotes the number of samples to be obtained from the negative set. This resulted to an equal number of positive and negative S, T, and Y fragments from the data sets as shown in Table In order to investigate the surrounding residues, with reference to KinasePhos ,16, sequidentity ,23 is emi and Aj, which surround the phosphorylation site. In order to extract motifs that have conserved biochemical property of amino acids, the twenty types of amino acids are categorized into five groups: neutral, acid, basic, aromatic, and imino groups, as shown in Additional File MDDLogo  was applmn Xrepresents the number of sequences that have the amino acids of group m in position Ai and have the amino acids of group n in position Aj, for each pair  with i≠j. mn Eis calculated as mR X= m1X+ ... +m5X, Cn X= 1nX+ ... +5nX, and X denotes the total number of sequences. If a strong dependence is detected  between two positions, then the process is continued as described by Burge and Karlin [where d Karlin . As the d Karlin . These wm × w elements was used to represent each residue of a training dataset, where m stands for the window size and w consists of 21 elements including 20 types of amino acids and one for terminal signal. Each MDDLogo-identified substrate motif contained a corresponding PWM with m × w elements, as illustrated in Figure A five-fold cross-validation evaluation was performed in order to determine which amino acid features were best utilized in establishing models that can effectively identify phosphorylation sites. Support vector machines (SVMs) were generated from the positive data and negative data of the training set. Based on binary classification, the concept behind SVM is to map the input samples into a higher dimensional space using a kernel function, and then to find a hyper-plane that discriminates between the two classes with maximal margin and minimal error. In this work, a public SVM library, LIBSVM , was empk-fold cross validation. In doing so, the training data was divided into k groups by splitting each dataset into k approximately equal sized subgroups. During cross-validation, one subgroup is regarded as the test set, and the remaining k-1 subgroups are regarded as the training set. The cross-validation process is repeated k rounds, with each of the k subgroups being used as a test set. The k results are then combined to produce a single estimation. The advantage of k-fold cross-validation is that all original data are regarded as both training set and test set, and each data is used for testing exactly once [k was set to five.Prior to the construction of a final model, the predictive performance of models using different parameters were evaluated by performing tly once . For thiThe following measures were used to gauge the predictive performance of the trained models: Sensitivity (Sn) = TP/(TP+FN), Specificity (Sp) = TN/(TN+FP), Accuracy  =  was used to represent each MDDLogo-identified substrate motif or Phospho.ELM kinase-specific motif. The measurement of Euclidean distance  was applAs shown in Additional File In order to further investigate the identified kinases, a literature survey was done. Reports have been published that CDK group, especially the CDK2, is involved in the transcription and replication of Human Immunodeficiency Virus - 1 by means of phosphorylation ,37. Prevhttp://csb.cse.yzu.edu.tw/ViralPhos/. As shown in Figure To aid in the analysis of virus phosphorylation, ViralPhos has been implemented as a web-based resource freely accessible at We have developed a novel method for identifying potential virus substrate site specificities and give information on its likely catalytic host kinase. We have identified informative motifs that matched with several well-studied kinase groups including CDK, MAPK, CK2, and PKB as potential catalytic kinases for virus protein substrates. A five-fold cross validation evaluation shows that the proposed method can identify virus phosphorylation sites based on the MDDLogo-identified motifs. Furthermore, an independent test done using data not included in the model training confirms the ability of our MDDLogo-clustered SVMs. The high sensitivity and specificity of MDDLogo-clustered SVMs show that the substrate site motifs are effective for the identification of potential viral protein phosphorylation sites. Overall, this study provides valuable information to the scientific community about what kind of host kinases may be responsible for the phosphorylation of viral proteins. However, it should be noted that the motif result is dependent on the experimentally verified virus phosphorylation sites used as a training data set. Future direction of this work would require the inclusion of a more abundant set of experimentally verified kinase-catalyzed virus phosphorylation sites.The authors declare that they have no competing interests.TYL conceived and supervised the project. KYH, CTL and NAB were responsible for the design, computational analyses, implemented the web-based tool, and drafted the manuscript with revisions provided by TYL and THC. All authors read and approved the final manuscript.http://csb.cse.yzu.edu.tw/ViralPhos/. All of the data set used in this work is also available for download in the website.ViralPhos can be accessed via a web interface, and is freely available to all interested users at Supplementary Table S1. Data resources of training set and independent testing setClick here for fileSupplementary Table S2. The amino acids group used in MDDLogo clusteringClick here for fileSupplementary Table S3. MDDLogo-identified motifs of virus phosphorylation dataClick here for fileSupplementary Table S4. Comparison of pSer and pThr motifs between MDDLogo and Motif-XClick here for fileSupplementary Table S5. Five-fold cross validation results on pSer MDDLogo-clustered SVM models trained with unbalanced positive and negative datasetsClick here for fileSupplementary Table S6. Five-fold cross validation results on pThr MDDLogo-clustered SVM models trained with unbalanced positive and negative datasetsClick here for fileSupplementary Table S7. Motif comparison between MDDLogo-clustered pSer virus motifs and well-studied kinase substrate motifsClick here for fileSupplementary Table S8. Motif comparison between MDDLogo-clustered pThr virus motifs and well-studied kinase substrate motifsClick here for file


Saurida elongata) muscle protein was hydrolyzed using neutral protease to produce protein hydrolysate (LFPH), and the hydrolysis conditions were investigated using response-surface methodology. The optimum conditions for producing peptides with the highest angiotensin-I converting enzyme (ACE)-inhibitory activity were the following: enzyme-to-substrate ratio of 10,000 U/g, temperature of 48 °C, pH 7.0, and hydrolysis time of 2 h. Under these conditions, the ACE-inhibitory activity of LFPH and the degree of hydrolysis were 84% and 24%, respectively. A novel ACE-inhibitory peptide was isolated from LFPH using ultrafiltration, Sephadex G-15, and high-performance liquid chromatography. The amino acid sequence of the ACE-inhibitory peptide was identified as Ser-Pro-Arg-Cys-Arg (SPRCR), and its IC50 was 41 ± 1 µM. Lizard fish ( Hypertension is considered to be the most common chronic disease and a major risk factor for cardiovascular disease, a main cause of death worldwide ,2. AngioN-terminal and C-terminal. Each domain is catalytically independent, and binding to zinc is crucial for enzymatic activity  × 100 (5)IP (%) = [1 − , respectively. IC50, the inhibitor concentration needed to inhibit 50% of enzyme activity, was determined by regression analysis of ACE inhibition (%) versus the log of the inhibitor concentration. where 1), E/S (X2), and pH (X3) were employed at five levels. The experimental designs are shown in In the present study, the CCD of the three factors was used to optimize the enzymatic hydrolysis conditions of lizard fish muscle protein. Temperature . A P-value of less than 0.05 was chosen for statistical significance.In the formula above (Equation 6), y  is the response variable; xLizard fish protein hydrolysate (LFPH) was ultrafiltrated with a 5000 MW cut-off filter . The fraction LFPH-І, which was able to pass through the 5 kDa membrane, was lyophilized and used for further separation.18 column  at a flow rate of 0.5 mL/min with a linear gradient of solvent B from 0% to 50% for 60 min. In the second HPLC step, the fractions showing the most efficient ACE-inhibitory activity were purified on a Hypersil ODS C18  with the gradient  at a flow rate of 0.5 mL/min. The fraction with the highest ACE-inhibitory activity was further purified with Zorbax SB C18  and eluted with the gradient  at a flow rate of 0.5 mL/min. The fraction exhibiting the highest ACE-inhibitory activity was collected, lyophilized, and used to identify the amino acid sequence.The fraction LFPH-І was fractionated using a Sephadex G-15 column , which had been previously equilibrated with distilled water. The column was eluted with water at a flow rate of 1 mL/min and the elution was monitored at 280 nm. The fraction with the highest ACE-inhibitory activity was collected, lyophilized, and then purified by three-step HPLC. Elution peaks were monitored at 220 nm. Solvent A was 0.1% (v/v) TFA in water, and solvent B was 0.1% (v/v) TFA in acetonitrile. In the first HPLC step, separations were performed on a Hypersil ODS CThe amino acid sequence was identified using a 4800 Plus MALDI TOF/TOF™ Analyzer . It was performed at the School of Life Sciences and Technology, Guangxi University, Nanning, China. The peptide was synthesized by GL Biochem Ltd, Shanghai, China.in vivo anti-hypertensive effects on animals. A CCD was used to estimate the effects of temperature, E/S, and pH on response and factor interactions. The optimum operating conditions for enzymatic hydrolysis to achieve maximum ACE-inhibitory activity were a temperature of 48 °C, pH of 7.0, and E/S of 10,000 U/g. Furthermore, a novel ACE-inhibitory peptide was purified using ultrafiltration, gel filtration, and HPLC. The ACE-inhibitory peptide was identified and the peptide with sequence SPRCR was synthesized to confirm the ACE inhibitory activity. Therefore, it is to be expected that this peptide could be applied as a drug for preventing hypertension. However, further studies are being carried out to confirm its


Vascular smooth muscle cells (VSMCs) of the arterial wall play a critical role in the development of occlusive vascular diseases. Cysteine-rich protein 2 (CRP2) is a VSMC-expressed LIM-only protein, which functionally limits VSMC migration and protects against pathological vascular remodeling. The multifunctional cytokine TGFβ has been implicated to play a role in the pathogenesis of atherosclerosis through numerous downstream signaling pathways. We showed previously that TGFβ upregulates CRP2 expression; however, the detailed signaling mechanisms remain unclear.TGFβ treatment of VSMCs activated both Smad2/3 and ATF2 phosphorylation. Individually knocking down Smad2/3 or ATF2 pathways with siRNA impaired the TGFβ induction of CRP2, indicating that both contribute to CRP2 expression. Inhibiting TβRI kinase activity by SB431542 or TβRI knockdown abolished Smad2/3 phosphorylation but did not alter ATF2 phosphorylation, indicating while Smad2/3 phosphorylation was TβRI-dependent ATF2 phosphorylation was independent of TβRI. Inhibiting Src kinase activity by SU6656 suppressed TGFβ-induced RhoA and ATF2 activation but not Smad2 phosphorylation. Blocking ROCK activity, the major downstream target of RhoA, abolished ATF2 phosphorylation and CRP2 induction but not Smad2 phosphorylation. Furthermore, JNK inhibition with SP600125 reduced TGFβ-induced ATF2 (but not Smad2) phosphorylation and CRP2 protein expression while ROCK inhibition blocked JNK activation. These results indicate that downstream of TβRII, Src family kinase-RhoA-ROCK-JNK signaling pathway mediates TβRI-independent ATF2 activation. Promoter analysis revealed that the TGFβ induction of CRP2 was mediated through the CRE and SBE promoter elements that were located in close proximity.Csrp2 promoter to cooperatively control CRP2 induction in VSMCs, which represents a previously unrecognized mechanism of VSMC gene induction by TGFβ.Our results demonstrate that two signaling pathways downstream of TGFβ converge on the CRE and SBE sites of the  Vascular smooth muscle cells (VSMCs) in the tunica media of the arteries play important roles in regulating blood pressure and vascular tone. In normal vessels, these VSMCs exhibit a quiescent and differentiated phenotype and express proteins involved in the contractile functions such as smooth muscle (SM) myosin heavy chain and SM α-actin. However, in contrast to striated muscle cells, adult VSMCs retain significant plasticity, known as phenotypic modulation. In response to arterial injury, VSMCs de-differentiate, downregulate SM marker genes, and change to a proliferative and migratory phenotype, leading to lesion formation and occlusive vascular disease. Thus, pCysteine-rich protein (CRP) 2, a LIM-only CRP family member,4. ImporThe multifunctional cytokine TGFβ contributes to the pathogenesis of atherosclerosis and restenosis,6. DurinCsrp2 (mouse CRP2 gene symbol)-deficient VSMC migration, demonstrating the functional importance of CRP2 induction by TGFβ in regulating VSMC migration[We identified in a previous study that TGFβ induces CRP2 expression in VSMCs and TGFβ treatment reduces wild type but not igration. TGFβ upigration; howeverTo investigate the signaling pathways that mediate CRP2 induction by TGFβ, we first examined type I TGFβ receptor (TβRI) downstream signaling. We pretreated VSMCs with vehicle or TβRI kinase inhibitor SB431542 for 30 min, followed by stimulation with or without TGFβ for 24 h and then examined CRP2 expression levels. SB431542 significantly reduced TGFβ-induced CRP2 expression  has been demonstrated to bind and present TGFβ to type II TGFβ receptor (TβRII), thereby increases type II receptor binding affinity and cell responsiveness to TGFβ. To deteWe next investigated how TβRII mediates ATF2 activation independent of TβRI in VSMCs. In JEG3 choriocarcinoma cells, Src kinase is involved in TGFβ-induced early signaling. In addiSince RhoA kinase (ROCK) is a major target of RhoA and that ROCK has been implicated in SMC differentiation by modulating TGFβ-Smad signaling, we examP < 0.05)  revealed that in addition to the previously identified CRE site at bp −461  elements in the 5′ promoter[We demonstrated that both CRE and SBE sites contribute to CRP2 upregulation by TGFβ Figure . Importacription. Interescription. TGFβ stcription. Since ber genes. For exapromoter,34. HoweCsrp2 promoter to cooperatively induce Csrp2 gene transcription and protein expression  to generate SBE681mut and SBE445mut constructs, respectively. The CREmut/SBE445mut construct with double mutations at –461CRE and –445SBE was generated using –795CREmutCsrp2-luc[The –795eviously and usedsrp2-luc as a temsrp2-luc was obtasrp2-luc. The consrp2-luc that conCsrp2-luc (0.33 μg/well) and 0.67 μg/well of expression plasmid  and luciferase activity measured 24 h later.Primary VSMCs were isolated from mouse aortas and cultured in Dulbecco’s modified Eagle’s medium (DMEM) as described. Cells o6 cells) with Gene Pulser Xcell™ Electroporation System (Bio-Rad) using 400 V, 10 ms, square wave parameters. Following recovery, cells were serum starved (0.5% FBS in DMEM) and total RNA or protein isolated at indicated time points for real time-PCR or Western blot analysis. For dominant-negative TβRII experiments, VSMCs were electroporated with pCMV5 vector or pCMV5 HA-TβRII(∆Cyt), serum starved, treated with or without TGFβ, and then total proteins isolated for Western blot analysis at different time points.For overexpression experiments, VSMCs were electroporated with vector control or expression plasmid Smad2, C2/ATF2, or Smad2 + C2/ATF2  for 24 h, cells were stimulated with or without TGFβ. Total proteins were prepared at the indicated time points using extraction buffer containing protease inhibitor Complete™ (Roche) and Halt™ Phosphatase Inhibitor Cocktail (Thermo Scientific) for Western blot analysis as described. To inhi71, #9221), phospho-Smad2 , phospho-Smad3 , Smad2/3 (#3102), phospho-JNK Thr183/Tyr185 (#9251), JNK (#9252), phospho-p38 Thr180/Tyr182 (#9211), p38 (#9212), phospho-ERK1/2 Thr202/Tyr204 (#9101), ERK1/2 (#9102), and TβRIII (#2519). ATF2(C19) antibody (Santa Cruz Biotechnology) was used to detect total ATF2. CRP2-(91–98) antiserum[To assess phosphorylated and total protein of signaling molecules, membranes were incubated with antibodies against respective phospho- or total protein. The following antibodies were purchased from Cell Signaling Technology: phospho-ATF2 (Thrntiserum was used5 cells) were plated on 60-mm dish and after overnight incubation, cells were transfected with 20 nM of negative control or target siRNA in Opti-MEM® I Reduced Serum Medium using Lipofectamine RNAiMAX as described by the manufacturer’s protocol (Life Technologies).To suppress mRNA levels of Smad2/3, ATF2, TAK1, TRAF6, RhoA, TβRI, TβRII, and TβRIII, we performed knockdown experiments with small interfering RNA (siRNA). The ON-TARGET plus SMART pool siRNAs and negative control siRNA were obtained from Dharmacon. VSMCs  were incubated with 400 μg of GST-Rhotekin-RBD and glutathione resin at 4 °C for 1 h. Following washing, bound Rho was eluted by SDS sample buffer. The eluted samples and the total cell lysates were then subjected to Western blot analysis with RhoA antibody (26C4)  to detect active and total RhoA, respectively.RhoA activation was assessed using an Active Rho Pull-Down and Detection Kit (Thermo Scientific) by immunoprecipitating GTP-bound RhoA with GST fusion protein of Rhotekin RhoA binding domain (GST-Rhotekin-RBD). Briefly, VSMCs plated in 150-mm dish were washed with ice-cold PBS and lysed in buffer containing 25 mM Tris pH 7.2, 150 mM NaCl, 5 mM MgClT method.Total RNA was isolated using an RNeasy Mini kit (QIAGEN) according to the manufacturer’s instructions. One μg RNA was first reverse transcribed to cDNA with random primers using SuperScript III reverse transcriptase (Invitrogen). Quantitative real time-PCR was performed with the transcribed cDNA and SYBR® FAST Universal 2X qPCR Master Mix (KAPA) in triplicates using the 7500 real time PCR system (Applied Biosystems) to detect CRP2 mRNA expression. The primers used for CRP2 are: forward, 5′-CTGACTGAGAAAGAAGGCGAAATC-3′ and reverse, 5′-TGCTGGCTGTTTCACAGTAGTGA-3′. β-actin was used as an internal control for normalization with the following primers: forward, 5′-GAGAGGTATCCTGACCCTGAAG-3′; reverse, 5′-TGATCTGGGTCATCTTTTCACGG-3′. Quantification was performed by the comparative Ct-test. P values <0.05 are considered statistically significant.Data are presented as mean ± S.E. of at least three independent experiments. All results were analyzed statistically by Student’s 6GG; CRE: Cyclic AMP response element; CRP2: Cysteine-rich protein 2; Csrp2: Mouse CRP2 gene symbol; ROCK: RhoA kinase; SBE: Smad binding element; siRNA: Small interfering RNA; SM: Smooth muscle; TβR: TGFβ receptor; TCE: TGFβ control element; VSMCs: Vascular smooth muscle cells.CArG element: CC(A/T)The authors declare no competing interests.MLW, CHC, YTL, YJJ, and YCH performed experiments, MLW, LTY, LC, MDL, and SFY analyzed and interpreted the data, MLW, MDL, and SFY prepared the figures. MLW, MDL, and SFY designed the research and wrote the manuscript. All authors had a final approval of the manuscript.


The kidney maintains water homeostasis by modulating aquaporin 2 (AQP2) on the plasma membrane of collecting duct principal cells in response to vasopressin (VP). VP mediated phosphorylation of AQP2 at serine 256 is critical for this effect. However, the role of phosphorylation of other serine residues in the AQP2 C-terminus is less well understood. Here, we examined the effect of phosphorylation of S256, S261 and S269 on AQP2 trafficking and association with recycling pathway markers. We used LLC-PK1 cells expressing AQP2(S-D) or (S-A) phospho mutants and a 20°C cold block, which allows endocytosis to continue, but prevents protein exit from the trans Golgi network (TGN), inducing formation of a perinuclear AQP2 patch. AQP2-S256D persists on the plasma membrane during cold block, while wild type AQP2, AQP2-S256A, S261A, S269A and S269D are internalized and accumulate in the patch. Development of this patch, a measure of AQP2 internalization, was most rapid with AQP2-S256A, and slowest with S261A and S269D. AQP2-S269D exhibited a biphasic internalization profile with a significant amount not internalized until 150 minutes of cold block. After rewarming to 37°C, wt AQP2, AQP2-S261A and AQP2-S269D rapidly redistributed throughout the cytoplasm within 20 minutes, whereas AQP2-S256A dissipated more slowly. Colocalization of AQP2 mutants with several key vesicular markers including clathrin, HSP70/HSC70, EEA, GM130 and Rab11 revealed no major differences. Overall, our data provide evidence supporting the role of S256 and S269 in the maintenance of AQP2 at the cell surface and reveal the dynamics of internalization and recycling of differentially phosphorylated AQP2 in cell culture. Aquaporin-2 (AQP2) is critical for the maintenance of systemic water balance in mammals. Apical accumulation of AQP2 in renal collecting duct principal cells increases the permeability of the epithelium, allowing water to flow down its osmotic gradient into the interstitium and enter the circulation Recently, various non-canonical (i.e. non-VP mediated) pathways for AQP2 membrane accumulation have been demonstrated by our group and others. Phosphorylation of the AQP2 COOH terminus can be regulated by activation of protein kinase G (PKG) in response to elevated cGMP Phospho-proteomic studies Therefore, in this study we set out to investigate the role of AQP2 phosphorylation sites on non-stimulated (constitutive recycling) endocytosis and exocytosis using AQP2 with point mutations that mimic either the phosphorylated or un-phosphorylated state of serine 256, 261 and 269. We are able to isolate the endocytotic and exocytotic pathways by employing the “cold block” and “cold block release” methods to follow AQP2 trafficking. As observed nearly twenty years ago, the transport of membrane proteins from the Golgi to the plasma membrane can be blocked by incubating cells at 20°C, inhibiting the exit of protein from the Golgi All chemicals unless otherwise noted were purchased from Sigma-Aldrich (St. Louis MO), and cell culture reagents were obtained from Invitrogen (Carlsbad CA). Monoclonal antibodies against c-myc were generated from the 9E10 hybridoma cell line, purchased from American Type Culture Collection . Secondary antibodies tagged with either Cy3, CY5.5 or FITC were obtained from Jackson Immunoresearch Laboratories (West Grove PA). The following commercial primary antibodies were purchased from their respective vendors: EEA1 , Rab5 , Rab7 , Rab10 (Sigma-Aldrich R8906), HSP70/HSC70 , Lysotracker Red (Invitrogen), Clathrin . LLC-PK1 cells were purchased from ATCC (CL-101).GCAGTGGAG 3′5′ GTGCGGCGGCAG, S256A: (R) CGTCACCTCGAGGTGAGA 3′5′ GCCGTC. S256D: (F) GACGTGGAG 3′5′ GTGCGGCGGCAG S256D: (R) CTGCACCTCGAGGTGAGA 35′ GCCGTC,′ S261A: (F) GCTCCTCAGAGCC 3′5′ GGTGGAGCTCCAC, S261A: (R) AGCGTGGAGCTCCACC 3′5′ GGCTCTGAGG. S269A: (F) GCCAAGGCCGAACAAAAGC 3′5′ GAGCCTGCCTGCCGGC, S269A: (R) GGCGCCGCGAGGCAGGCTC 3′5′ GCTTTTGTTCGGCCTT. S269D: (F) GACAAGGCCGAACAAAAGC 3′5′ GAGCCTGCCTGCCGGC, S269D: (R) GTCGCCGCGAGGCAGGCTC 3′5′ GCTTTTGTTCGGCCTT. Stable cell lines expressing these phospho-mimic AQP2 mutants were obtained by transfection of the porcine kidney cell line, LLC-PK1, using Lipofectamine (Invitrogen) and selection under 500 µg/mL G418. All cell lines were maintained in DMEM supplemented with 10% bovine serum and 1% penicillin/streptomycin at 37°C in a humidified 5% CO2 atmosphere.Point mutations substituting either an aspartic acid (D) or alanine (A) for serine (S) on the C-terminus of AQP2 at positions 256, 261 and 269 were generated using site directed mutagenesis (Invitrogen) as previously reported LLC-PK1 - AQP2 cells were plated on 15×15 mm glass cover slips (Electron Microscopy Sciences) at least 24 hours before experimentation. To inhibit newly synthesized protein production, cycloheximide was added to the culture medium (10 µg/mL) for 60 minutes prior to, and maintained in the medium during cold block. Cold block was performed by placing the culture plates at 20°C in a water bath for various time points up to 150 minutes. Cells were then fixed with 4% paraformaldehyde/PBS (PH 7.4) and subjected to immunofluorescence staining. For the cold block and release experiment, after cold block for 2 hours, which gives the maximal perinuclear patch structure, cells were brought to 37°C to allow protein/vesicle recycling to occur. Cells were harvested at various time points after cold block release, fixed and processed for immunofluorescence staining.AQP2 and subcellular markers were localized by immunofluorescence staining via a standard protocol. Cells were permeabilized in 0.01% Triton X-100 in PBS for 4 minutes, blocked with a 1% BSA/PBS for 20 minutes and then incubated with primary antibody overnight at 4°C. After washing with PBS, cells were incubated with the secondary antibodies at room temperature for one hour. Subsequently, the cover slips were washed and mounted in Vectashield containing DAPI  and visualized on a Nikon 80i microscope with a ORCA95 camera . For quantitative analysis of AQP2 trafficking, all cover slips were stained under the same conditions and imaged with the same microscope parameters. Analysis of the AQP2 patch fluorescence was performed using IPLAB software . The region of interest used to measure AQP2 patch fluorescence was determined by applying an intensity threshold to the visible perinuclear accumulation of AQP2 . ColocalStatistics were performed with the Prism software . Data for each treatment (cold block or cold block release) were first compared for significance with a one-way ANOVA. Differences in means were then compared between AQP2 mutants at each time point using the student's T-test (two tailed). Each experiment was repeated at least three times. Statistical significance was determined at a p value<0.05.Stable expression of AQP2 phosphorylation mutants containing a c-myc tag fused to the COOH terminus was achieved in LLC-PK1 cells. Based on the rat AQP2 sequence, point mutations at serine residues 256, 261, or 269 were created substituting either aspartic acid (D), mimicking the charged state of phosphorylation, or alanine (A), which mimics the de-phosphorylated state . AQP2 expression in these cell lines was confirmed by western blot using an antibody against the c-myc tag and a commercially available polyclonal goat anti-AQP2 antibody recognizing AQP2 C-terminus . Both antibodies showed similar results and the immunoblot using anti-c-myc antibody is presented here .de novo protein synthesis with cycloheximide in the culture medium. Cycloheximide at this concentration has been previously shown by our group to significantly inhibit AQP2 and V2R synthesis To study the role of phosphorylation of AQP2 on its endocytosis, LLC-PK1 cells expressing the AQP2 constructs were incubated at 20°C (cold block) for up to 150 minutes and evaluated by immunofluorescence staining with anti-c-myc antibodies as previously described To quantify the rate of accumulation of endocytosed AQP2, the formation of the perinuclear patch was evaluated by immunofluorescence staining every 30 minutes over 150 minutes of cold block at 20°C. Representative images for each AQP2 mutant at 0, 45 and 120 minutes of cold block are displayed in To investigate the impact of the phosphorylation mutations on the exocytosis of AQP2, cells were returned to 37°C after 120 minutes of cold block at 20°C. In AQP2 is a constitutively trafficked protein that is recycled from the plasma membrane beginning in clathrin coated pits In Cold block of LLC-PK1 cells inhibits protein exit from the TGN and results in a condensation of AQP2 staining in a perinuclear region that is also labeled by the TGN protein GM130 . In agreAssociation with EEA1 decreased during the cold block, with a subsequent increase following cold block release . Rab7- AIn vivo, VP stimulation leads to dramatic apical membrane staining of AQP2 with antibodies specific for phosphorylation at serine 256 (pS256), and semi-quantitative analysis by western blot reveals an increase in the percentage AQP2 with S256 phosphorylation. In vitro, the dominant role of S256 in membrane accumulation is supported by the constitutive membrane presence of AQP2 in cells expressing the phospho-mimic mutation AQP2-S256D, an effect that is not altered by S-D or S-A substitutions at S261, or S269 in vitro by ubiquitination at K270 following the addition of 12-tetradecanoylphorbol-13-acetate (TPA) The current model of regulated AQP2 trafficking links VP stimulated phosphorylation of serine 256 with membrane accumulation of the water channel In contrast, AQP2-S256A remains mostly cytosolic even during stimulation of cells by VP and/or forskolin. The S256A mutation, however, does not prevent the constitutive recycling of the water channel, and membrane accumulation of AQP2 S256A can be readily observed upon acute inhibition of endocytosis We quantified some significant differences among the AQP2 mutants in the rate at which the perinuclear patch accumulated. The most rapid accumulations were seen with the S256A and S261A mutants. This is consistent with the fact that the S256A mutant cannot be phosphorylated at the critical S256 site, and its transit through the plasma membrane is not retarded, as could be the case for at least some of the wild type protein, which may have some level of constitutive S256 phosphorylation. The greater speed of S261A accumulation compared to the wild type protein is more difficult to rationalize, since it too could have some baseline level of S256 phosphorylation. Our data indicate that, at the very least, VP-induced dephosphorylation of S261 is not by itself a signal for cell surface retention of AQP2.in vivo, and a role in retarding endocytosis has been suggested in vivo, antibodies against pS269 label only the plasma membrane The role of the S269 site is slowly emerging. pS269 is observed only on the apical membrane in vivo remains to be determined.Upon re-warming of cells to 37°C, the tight perinuclear patch containing AQP2 begins to disperse, as material is released from the cold-induced TGN export block. It has been shown for AQP2, as well as in other systems, that this released material can enter the secretory pathway, and be inserted into the plasma membrane through constitutive vesicle exocytosis While the differential role of various AQP2 phosphorylation sites on AQP2 regulated trafficking as well as constitutive recycling will continue to be the subject of future studies, our data demonstrate that differential phosphorylation of AQP2 affects the rate and pattern of endocytosis and recycling of AQP2 in cells.Figure S1Region of Interest around perinuclear patch. The region of interest (yellow line) used to measure AQP2 patch fluorescence was determined by applying an intensity threshold to the visible perinuclear accumulation of AQP2.(TIF)Click here for additional data file.Figure S2Quantification of colocalization between AQP2 and markers of the subcellular compartment. The fraction of AQP2 signal colocalized with each subcellular compartment marker is presented for 0, 45, and 120 minutes of cold block as well as for 30 minutes following cold block release. For each data set, the colocalizations were calculated from multiple individual cells in confocal microscope stacks taken at a step size of 1 µm. Means are presented with standard error bars. Despite the formation of a tight perinuclear patch in the same vicinity as the Golgi apparatus, as can be observed in (PDF)Click here for additional data file.


StudiesFoxO, as the transcription factor of rictor, plays a key role in the assembly of mTORC2; FoxO also inhibits mTORC1, but the inhibition depends on expression of Sestrin3, rictor, and activation of AMPK . FoxO3a Warburg’s hypothesis of aerobic glycolysis centers on the respiratory damage  as the primary cause of malignancy. This, in our opinion, is an aberration of mitochondrial function. Traditionally mitochondria had been viewed as the ATP producing “power house” of the cell. It should be recognized that ATP production is only one of the short time functions of mitochondria. Substrate shuttles like, malate, aspartate, glutamate and citrate are critical for anaplerotic and cataplerotic reactions of Krebs cycle. They are the key sources of carbon and nitrogen requirements for protein and lipid biosynthesis of growing cells , 6. ThesGlutamine, avidly consumed by actively proliferating cancer cells , 8, has Glutaminase, which hydrolyzes glutamine to glutamate and ammonia, is up-regulated in several cancer cells , 49. Sil+ co-transport (Glutamine and glutamate are the substrates for leucine and cystine antiporters, while leucine is an activator of anabolism , cystineransport  promotesransport . In contransport .Production of lactic acid in cancer cells is one of the strong arguments in favor of aerobic glycolysis . Lactate+ dependent forms, and the mitochondrial NAD+ dependent form (Mitochondrial substrate shuttles, like aspartate, glutamate, and succinate, which are key source of NADPH required for biosynthesis and the ATP, are also sources for malate ent form , 68. ME ent form . Inhibitent form . Anaplerent form ; transamIn summary, we suggest that nitrogen source is critical to cell growth and oxygen’s role is to activate ATP production and limit glucose uptake. Nitrogen uptake and reduced dependency on glucose for growth in the presence of oxygen is the “Pasteur’s dictum.” Activation of mTORC1 by amino acids, independent of IIS, is the molecular recognition of “Pasteur’s dictum.” Activation of mTORC1 appears to precede mTORC2, which perhaps facilitates translation of Akt and its subsequent S473 phosphorylation and promotes glucose uptake and energy production as well as IIS activation. Stabilization of mTORC1 by IIS for anabolic activity of cells depends on ATP/ADP ratio in the absence of which, cells may recycle as stem cells. In anabolic environment, mitochondria reprogram their function for biosynthetic activity to replenish the carbon and amino acid resources for lipid and protein biosynthesis. Inhibition of mTORC1 in growing cells depends on ROS and AMPK. Higher levels of dietary amino acids result in longtime mTORC1 activation and inhibition of mTORC2 and insulin resistance, which appear to be the cause of metabolic pathologies and the secret of healthy life under dietary restriction .


We could recently demonstrate that the predominant NBC isoform in murine colonic crypts is electrogenic NBCe1-B, and that secretagogues cause NBCe1 exocytosis, which likely represents a component of NBC activation. Since protein kinase C (PKC) plays a key role in the regulation of ion transport by trafficking events, we asked whether it is also involved in the observed NBC activity increase. Crypts were isolated from murine proximal colon to assess PKC activation as well as NBC function and membrane abundance using fluorometric pHi measurements and cell surface biotinylation, respectively. PKC isoform translocation and phosphorylation occurred in response to PMA-, as well as secretagogue stimulation. The conventional and novel PKC inhibitors Gö6976 or Gö6850 did not alter NBC function or surface expression by themselves, but stimulation with forskolin (10−5 M) or carbachol (10−4 M) in their presence led to a significant decrease in NBC-mediated proton flux, and biotinylated NBCe1. Our data thus indicate that secretagogues lead to PKC translocation and phosphorylation in murine colonic crypts, and that PKC is necessary for the increase in NBC transport rate and membrane abundance caused by cholinergic and cAMP-dependent stimuli.The colonic mucosa actively secretes HCO In the latter, the relevance of PKC in regulating basolateral base-loading transporters like NBC is largely unknown.In renal cells, early functional data indicates a stimulatory effect of PKC on NaWe could previously demonstrate that cholinergic stimulation of NBC in isolated murine colonic crypts is partially reversible by treatment with PKC inhibitors The polyclonal anti-NBCe1 antibody K1A directed against the cytoplasmic COOH-terminus common to the NBCe1-A and NBCe1-B subtypes was generously supplied by Walter Boron ad libitum, and were used for the experiments at the age of 3 months. All experiments followed approved protocols and guidelines from the Medical School of Hannover (Permit Number: AZ 2012/10) and the local authorities for the regulation of animal welfare (Niedersächsisches Landesministerium für Verbraucherschutz und Lebensmittelsicherheit).C57BL/6 mice were kept in the animal facility of Hannover Medical School under standardized light and climate conditions, had access to water and chow 2 narcosis, mice were sacrificed by cervical dislocation , keeping CO2 exposure as short as possible to minimize the effect on acid/base transporters. Subsequently, crypts were isolated as previously described 2, 5 sodium pyruvate, 10 HEPES, 5 EDTA, 1% BSA, and 5 glucose, pH 7.4, gassed with 100% O2) at 37°C for 12 min, harvested after gentle agitation, immersed in ice cold buffer A and stored on ice until use.After COi) was measured in BCECF-loaded crypts using a video imaging system exactly as previously described 4+-prepulse protocol by subsequent perfusion with buffers C, D and B (see i recovery rates were measured in the presence of the intended compounds and 700 μM dimethylamiloride (DMA), which we have shown to entirely inhibit Na+/H+ exchange activity in murine colonic crypts. Calibration of the 440- to 490-nm ratio was performed using the high K+-nigericin method Intracellular pH  and treated with PMA, secretagogues and/or PKC inhibitors according to the experimental protocol, incubated twice with biotinylation buffer , washed and lysed as previously described A previously validated amount of each fraction yielding signals within a linear range of optical density suitable for semi-quantification was loaded, and size fractionated as described by Hillesheim et al. http://rsb.info.nih.gov/ij/).To estimate the distribution of PKC isoforms in relation to NBCe1, the mid colon of C57B/6 mice was prepared. NBCe1 was visualized on cryosections stained with the K1A-antibody exactly as previously described i slope after the re-addition of Na+, which was determined by regression analysis, with the total buffering capacity at the initial pHi, including the intrinsic buffering capacity (βi) and, in addition, the CO2-dependent buffering capacity for CO2/HCO3−-containing solutions. Student's t-test in its paired and unpaired form, where appropriate, was used for pair-wise tests, and ANOVA was used for multiple comparisons (ANOVA for correlated samples in the biotinylation experiments), and Tukey's HSD (Honestly Significant Difference) Test as a post-hoc test. A p-value less than 0.05 was considered statistically significant.Results are given as means ± SE. Proton fluxes were calculated by multiplying the initial steep pH+/HCO3− cotransporter (NBC), we first asked how PKC isoforms are subcellularly localized in murine colon, and whether PKC activation by forskolin and carbachol occurs in isolated crypts. To this end, we stained murine colonic sections with antibodies against NBCe1 as well as conventional and novel PKC isoforms . As observed previously, NBCe1 was found in the basolateral membrane, with some signal in the cytoplasm likely representing inactive NBCe1 in the resting state In this study designed to investigate the role of PKC during secretagogue-associated activation of NaPKC activation occurs in parallel with its translocation to the membrane, and we thus performed experiments to verify whether this can be observed in our model. In a first approach, cell surface biotinylation with a general PKC antibody was carried out . In a se+/HCO3− cotransporter activity was fluorometrically determined in isolated murine colonic crypts +/H+ exchange (NHE) was pharmacologically separated using a dose of di-methyl-amiloride which is inhibitory for all NHE isoforms present in the colon Next, we sought to investigate the role of PKC during secretagogue-dependent NBC regulation in colonic crypts. We had previously shown that carbachol-induced NBC activation is abolished by Gö6850 and Gö6976, with a non-significant trend towards a weaker effect of Gö6976 Since NBC is regulated by membrane trafficking +/HCO3− cotransport is time-dependent and complex, and its investigation in various experimental systems has yielded controversial results. In the case of renal NBC, e. g., phorbol ester exerts a stimulatory effect in renal cells Xenopus oocytesThe effect of PKC on NaGiven the stimulatory effect of secretagogues on colonic NBC, we first investigated PKC translocation, which was enhanced, and phosphorylation by these compounds. Interestingly, only the phosphorylation of PKC-α, but not of PKC-ε induced by secretagogues was sensitive to PKC inhibition. This may be explained by several published observations: The phosphorylation of PKC-ε appears to be more complex, with different PKC isoenzymes involved, and the phosphorylation state of conventional vs. novel PKC differentially contributes to its degradation probability + and HCO3− dependent, DMA-insensitive pHi recovery we measure in crypts after imposing an acid load is mediated – at least predominantly – by NBCe1-B 49 is important for cAMP-dependent stimulation of NBCe1-B 3− secretion in pancreatic ducts in vivoHaving found that not only the known PKC activator carbachol, but also cAMP-dependent stimulation with forskolin can lead to PKC translocation and phosphorylation, we next fluorometrically assessed NBC activity after exposure to forskolin and/or PKC inhibitors. NBCe1-B is expressed at significant levels in murine colon +/K+/2Cl−-cotransporter − destined for secretion. In the colonic epithelial cell line T84, phorbol ester down-regulates basolateral Na+/K+/2Cl− cotransporter Given the importance of subcellular redistribution for NBC regulation In two elegant studies, the group of Irina Gritchtchenko delineated cholinergic regulation of NBC subtypes transfected into a salivary gland cell line, and the role of PKC in this process 3− transporters are relevant. NBCn1, which is essential for duodenal acid-base-balance +/HCO3− cotransporter in the murine colonic crypt, and there is no clear indication so far that the importance of other Na+/HCO3− transporters comes close. However, we cannot entirely exclude a functional role of other Na+/HCO3− transporters in our observations.One important question is whether other HCOXenopus oocytes, without changing its membrane abundance 38 and Ser65How exactly PKC exerts its regulatory effects on NBCe1 on a molecular level remains to be clarified. The question arises whether PKC acts exclusively via NBC endocytosis, or exerts an additional direct effect on the transporter. In salivary ParC5 cells, electrogenic NBC was also endocytosed by PMA Table S1Buffers used for the fluorometric experiments. All buffers contained a combined buffering system including HEPES/TRIS. Osmolarity was 290–300 mOsm/l, and pH was adjusted to 7.4. TMA: tetramethylammonium, HEPES: (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), TRIS: Tris(hydroxymethyl) –aminomethane.(DOC)Click here for additional data file.