Analysis of Inflammatory Cytokines in Postoperative Fontan Pleural Drainage.

Prolonged pleural drainage is a common complication in patients after Fontan palliation and is associated with short- and long- term morbidities. Among many potential etiologies, prolonged drainage has an inflammatory component, but there are no descriptions of cytokines in Fontan pleural drainage to date. This study aimed to examine the inflammatory make-up of Fontan pleural drainage. This prospective age-range-matched cohort study recruited 25 patients undergoing Fontan procedure and 15 bi-ventricular patients undergoing cardiopulmonary bypass (CPB). Chest tube samples were taken on postoperative day (POD) 1-4, 7, and 10. Cytokines were measured using Bio-Plex Assays. Univariate comparisons were made in patient characteristics and cytokine levels. Median age was 3.7 y (IQR 2.8-3.9) for controls and 2.5 y (IQR 2.1-2.9) in Fontan patients (p = 0.02). Median drainage duration and daily volume was higher in Fontan patients (both p < 0.001). Inflammatory cytokines (IL-17A, IFN-y, MIP-1ß, and TNF-α) were higher in Fontan patients than controls (all p < 0.02). There was an increase in pro-inflammatory cytokines (IL-8, MIP-1ß, and TNF-α) from POD1 to the last chest tube day (LCD) in Fontan patients (all p < 0.0001) and a decrease in the anti-inflammatory cytokine IL-10 (p = 0.001). There was no difference in cytokine concentration from POD1 to LCD among controls. There was a significant association with the cytokine concentration of TNF-α on POD1 and duration of chest tube drainage (p < 0.05). Inflammatory cytokine levels in the pleural fluid of Fontan patients are higher compared to bi-ventricular controls and rise over time where controls do not. This suggests ongoing localized inflammation that is not a result of CPB alone and may be an important contributor to pleural drainage in patients after the Fontan procedure.

Severe Acute Kidney Injury Following Stage 1 Norwood Palliation: Effect on Outcomes and Risk of Severe Acute Kidney Injury at Subsequent Surgical Stages.

OBJECTIVES: To identify associations of severe acute kidney injury early after stage 1 (Norwood) operation with risk of severe acute kidney injury and comorbidities at subsequent palliative stages in patients with hypoplastic left heart syndrome and other single ventricle lesions with left-sided obstruction. DESIGN: Retrospective cohort study. Severe acute kidney injury defined as Kidney Disease Improving Global Outcomes stage 3. SETTING: Single pediatric cardiac center. PATIENTS: Infants less than or equal to 28 days old with single ventricle physiology and left-sided obstruction undergoing stage 1 operation between September 2007 and November 2012 (n = 136). INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The occurrence rate of severe acute kidney injury was 21% (28/136) following stage 1, 12% (12/98) following stage 2 palliation (superior cavo-pulmonary anastomosis), and 10% (7/73) following stage 3 palliation (total cavo-pulmonary anastomosis). Severe acute kidney injury early after stage 1 operation was significantly associated with continuous intravenous loop diuretic infusion, need for extracorporeal membrane oxygenation, and in-hospital death (all p < 0.05). Gestational age at birth was associated with severe acute kidney injury at stage 2 (p = 0.04) and stage 3 (p = 0.01). Severe acute kidney injury at stage 1 was an independent risk factor for severe acute kidney injury at stage 2 (adjusted odds ratio, 4.3; 95% CI, 1.1-16.9; p = 0.04). Development of severe acute kidney injury after stage 1 was associated with longer mechanical ventilation time after stage 3 (p = 0.047). CONCLUSIONS: Severe acute kidney injury after stage 1 palliation was an independent risk factor for developing severe acute kidney injury at stage 2, and was associated with prolonged duration of mechanical ventilation following stage 3. Information on the incidence and associated risk factors for postoperative acute kidney injury in hypoplastic left heart syndrome patients from multiple congenital heart centers is a necessary next step to further understand the long-term burden of severe acute kidney injury after staged palliation.

Thioredoxin targets fundamental processes in a methane-producing archaeon, Methanocaldococcus jannaschii.

Thioredoxin (Trx), a small redox protein, controls multiple processes in eukaryotes and bacteria by changing the thiol redox status of selected proteins. The function of Trx in archaea is, however, unexplored. To help fill this gap, we have investigated this aspect in methanarchaea--strict anaerobes that produce methane, a fuel and greenhouse gas. Bioinformatic analyses suggested that Trx is nearly universal in methanogens. Ancient methanogens that produce methane almost exclusively from H2 plus CO2 carried approximately two Trx homologs, whereas nutritionally versatile members possessed four to eight. Due to its simplicity, we studied the Trx system of Methanocaldococcus jannaschii--a deeply rooted hyperthermophilic methanogen growing only on H2 plus CO2. The organism carried two Trx homologs, canonical Trx1 that reduced insulin and accepted electrons from Escherichia coli thioredoxin reductase and atypical Trx2. Proteomic analyses with air-oxidized extracts treated with reduced Trx1 revealed 152 potential targets representing a range of processes--including methanogenesis, biosynthesis, transcription, translation, and oxidative response. In enzyme assays, Trx1 activated two selected targets following partial deactivation by O2, validating proteomics observations: methylenetetrahydromethanopterin dehydrogenase, a methanogenesis enzyme, and sulfite reductase, a detoxification enzyme. The results suggest that Trx assists methanogens in combating oxidative stress and synchronizing metabolic activities with availability of reductant, making it a critical factor in the global carbon cycle and methane emission. Because methanogenesis developed before the oxygenation of Earth, it seems possible that Trx functioned originally in metabolic regulation independently of O2, thus raising the question whether a complex biological system of this type evolved at least 2.5 billion years ago.

A membrane-associated thioredoxin required for plant growth moves from cell to cell, suggestive of a role in intercellular communication.

Thioredoxins (Trxs) are small ubiquitous regulatory disulfide proteins. Plants have an unusually complex complement of Trxs composed of six well-defined types (Trxs f, m, x, y, h, and o) that reside in different cell compartments and function in an array of processes. The extraplastidic h type consists of multiple members that in general have resisted isolation of a specific phenotype. In analyzing mutant lines in Arabidopsis thaliana, we identified a phenotype of dwarf plants with short roots and small yellowish leaves for AtTrx h9 (henceforth, Trx h9), a member of the Arabidopsis Trx h family. Trx h9 was found to be associated with the plasma membrane and to move from cell to cell. Controls conducted in conjunction with the localization of Trx h9 uncovered another h-type Trx in mitochondria (Trx h2) and a Trx in plastids earlier described as a cytosolic form in tomato. Analysis of Trx h9 revealed a 17-amino acid N-terminal extension in which the second Gly (Gly(2)) and fourth cysteine (Cys(4)) were highly conserved. Mutagenesis experiments demonstrated that Gly(2) was required for membrane binding, possibly via myristoylation. Both Gly(2) and Cys(4) were needed for movement, the latter seemingly for protein structure and palmitoylation. A three-dimensional model was consistent with these predictions as well as with earlier evidence showing that a poplar ortholog is reduced by a glutaredoxin rather than NADP-thioredoxin reductase. In demonstrating the membrane location and intercellular mobility of Trx h9, the present results extend the known boundaries of Trx and suggest a role in cell-to-cell communication.

The level of expression of thioredoxin is linked to fundamental properties and applications of wheat seeds.

Work with cereals (barley and wheat) and a legume (Medicago truncatula) has established thioredoxin h (Trx h) as a central regulatory protein of seeds. Trx h acts by reducing disulfide (S-S) groups of diverse seed proteins (storage proteins, enzymes, and enzyme inhibitors), thereby facilitating germination. Early in vitro protein studies were complemented with experiments in which barley seeds with Trx h overexpressed in the endosperm showed accelerated germination and early or enhanced expression of associated enzymes (alpha-amylase and pullulanase). The current study extends the transgenic work to wheat. Two approaches were followed to alter the expression of Trx h genes in the endosperm: (1) a hordein promoter and its protein body targeting sequence led to overexpression of Trx h5, and (2) an antisense construct of Trx h9 resulted in cytosolic underexpression of that gene (Arabidopsis designation). Underexpression of Trx h9 led to effects opposite to those observed for overexpression Trx h5 in barley-retardation of germination and delayed or reduced expression of associated enzymes. Similar enzyme changes were observed in developing seeds. The wheat lines with underexpressed Trx showed delayed preharvest sprouting when grown in the greenhouse or field without a decrease in final yield. Wheat with overexpressed Trx h5 showed changes commensurate with earlier in vitro work: increased solubility of disulfide proteins and lower allergenicity of the gliadin fraction. The results are further evidence that the level of Trx h in cereal endosperm determines fundamental properties as well as potential applications of the seed.

Sex differences in thrombosis in mice are mediated by sex-specific growth hormone secretion patterns.

Sex differences in thrombosis are well described, but their underlying mechanism(s) are not completely understood. Coagulation proteins are synthesized in the liver, and liver gene expression is sex specific and depends on sex differences in growth hormone (GH) secretion--males secrete GH in a pulsatile fashion, while females secrete GH continuously. Accordingly, we tested the hypothesis that sex-specific GH secretion patterns cause sex differences in thrombosis. Male mice were more susceptible to thrombosis than females in the thromboplastin-induced pulmonary embolism model and showed shorter clotting times ex vivo. GH-deficient little (lit) mice were protected from thrombosis, and pulsatile GH given to lit mice restored the male clotting phenotype. Moreover, pulsatile GH administration resulted in a male clotting phenotype in control female mice, while continuous GH caused a female clotting phenotype in control male mice. Expression of the coagulation inhibitors Proc, Serpinc1, Serpind1, and Serpina5 were strongly modulated by sex-specific GH patterns, and GH modulated resistance to activated protein C. These results reveal what we believe to be a novel mechanism whereby sex-specific GH patterns mediate sex differences in thrombosis through coordinated changes in the expression of coagulation inhibitor genes in the liver.

Proteomics uncovers a role for redox in drought tolerance in wheat.

Proteomic analysis offers a new approach to identify a broad spectrum of genes that are expressed in living systems. We applied a proteomic approach to study changes in wheat grain in response to drought, a major environmental parameter adversely affecting development and crop yield. Three wheat genotypes differing in genetic background were cultivated in field under well-watered and drought conditions by following a randomized complete block design with four replications. The overall effect of drought was highly significant as determined by grain yield and total dry matter. About 650 spots were reproducibly detected and analyzed on 2-DE gels. Of these, 121 proteins showed significant change under drought condition in at least one of the genotypes. Mass spectrometry analysis using MALDI-TOF/TOF led to the identification of 57 proteins. Two-thirds of identified proteins were thioredoxin (Trx) targets, in accordance with the link between drought and oxidative stress. Further, because of contrasting changes in the tolerant and susceptible genotypes studied, several proteins emerge as key participants in the drought response. In addition to providing new information on the response to water deprivation, the present study offers opportunities to pursue the breeding of wheat with enhanced drought tolerance using identified candidate genetic markers. The 2-DE database of wheat seed proteins is available for public access at http://www.proteome.ir.

A redox-active FKBP-type immunophilin functions in accumulation of the photosystem II supercomplex in Arabidopsis thaliana.

Photosystem II (PSII) catalyzes the first of two photosynthetic reactions that convert sunlight into chemical energy. Native PSII is a supercomplex consisting of core and light-harvesting chlorophyll proteins. Although the structure of PSII has been resolved by x-ray crystallography, the mechanism underlying its assembly is poorly understood. Here, we report that an immunophilin of the chloroplast thylakoid lumen is required for accumulation of the PSII supercomplex in Arabidopsis thaliana. The immunophilin, FKBP20-2, belongs to the FK-506 binding protein (FKBP) subfamily that functions as peptidyl-prolyl isomerases (PPIases) in protein folding. FKBP20-2 has a unique pair of cysteines at the C terminus and was found to be reduced by thioredoxin (Trx) (itself reduced by NADPH by means of NADP-Trx reductase). The FKBP20-2 protein, which contains only two of the five amino acids required for catalysis, showed a low level of PPIase activity that was unaffected on reduction by Trx. Genetic disruption of the FKBP20-2 gene resulted in reduced plant growth, consistent with the observed lower rate of PSII activity determined by fluorescence (using leaves) and oxygen evolution (using isolated chloroplasts). Analysis of isolated thylakoid membranes with blue native gels and immunoblots showed that accumulation of the PSII supercomplex was compromised in mutant plants, whereas the levels of monomer and dimer building blocks were elevated compared with WT. The results provide evidence that FKBP20-2 participates specifically in the accumulation of the PSII supercomplex in the chloroplast thylakoid lumen by means of a mechanism that has yet to be determined.

Developmental changes in the metabolic protein profiles of wheat endosperm.

A combined two-dimensional gel electrophoresis-mass spectrometry approach was utilized to identify over 250 proteins of wheat (Triticum aestivum L., cv. Butte 86) starchy endosperm that participate in 13 biochemical processes: ATP interconversion reactions, carbohydrate metabolism, cell division, cytoskeleton, lipid metabolism, nitrogen metabolism, protein synthesis/assembly, protein turnover, signal transduction, protein storage, stress/defense, transcription/translation, and transport. Endosperm protein populations were compared at early (10 days post-anthesis, dpa) and late (36 dpa) stages of grain development. Analysis of protein number and spot volume revealed that carbohydrate metabolism, transcription/translation, and protein synthesis/assembly were the principal endosperm functions at 10 dpa followed by nitrogen metabolism, protein turnover, cytoskeleton, cell division, signal transduction, and lipid metabolism. Carbohydrate metabolism and protein synthesis/assembly were also major functions at 36 dpa, but stress/defense and storage were predominant. The results provide insight into biochemical events taking place during wheat grain development and highlight the value of proteomics in characterizing complex biochemical processes. Further, the proteome maps will facilitate future studies addressing the effects of genetic and environmental factors on the development and quality of wheat grain.

Thioredoxin targets of developing wheat seeds identified by complementary proteomic approaches.

The role of thioredoxin in wheat starchy endosperm was investigated utilizing two proteomic approaches. Thioredoxin targets were isolated from total KCl-soluble extracts of endosperm and flour and separated by 2-DE following (1) reduction of the extract by the NADP/thioredoxin system and labeling the newly generated sulfhydryl (SH) groups with monobromobimane (mBBr), and, in parallel, (2) trapping covalently interacting proteins on an affinity column prepared with mutant thioredoxin h in which one of the active site cysteines was replaced by serine. The two procedures were complementary: of the total targets, one-third were observed with both procedures and one-third were unique to each. Altogether 68 potential targets were identified; almost all containing conserved cysteines. In addition to confirming known interacting proteins, we identified 40 potential thioredoxin targets not previously described in seeds. A comparison of the results obtained with young endosperm (isolated 10 days after flowering) to those with mature endosperm (isolated 36 days after flowering) revealed a unique set of proteins functional in processes characteristic of each developmental stage. Flour contained 36 thioredoxin targets, most of which have been found in the isolated developing endosperm.

Thioredoxin reduction alters the solubility of proteins of wheat starchy endosperm: an early event in cereal germination.

A KCl-soluble, albumin/globulin fraction of wheat (Triticum aestivum L.) starchy endosperm was further separated into a methanol-insoluble fraction that contained metabolic proteins and a methanol-soluble fraction that contained "chloroform-methanol" or CM-like proteins. Reduction of the disulfide bonds of the CM proteins with thioredoxin or dithiothreitol altered their properties so that, like the metabolic proteins, they were insoluble in methanol. Glutathione had little effect, indicating dithiol specificity. Proteomic analysis of the CM protein fraction revealed the presence of isoforms of low molecular weight disulfide proteins (alpha-amylase, alpha-amylase/trypsin and WCI proteinase inhibitors, lipid transfer proteins, gamma-thionins), stress enzymes (Cu-Zn superoxide dismutase and peroxidase), storage proteins (alpha-, gamma- and omega-gliadins, low molecular weight glutenin subunits and globulins of the avenin N9 type), and a component of protein degradation (polyubiquitin). These findings support the view that, in addition to modifying activity and increasing protease sensitivity, reduction by thioredoxin alters protein solubility, thereby promoting processes of the grain starchy endosperm, notably the mobilization of reserves during germination and seedling development.

Thioredoxin h overexpressed in barley seeds enhances selenite resistance and uptake during germination and early seedling development.

The uptake, distribution and metabolism of selenite were examined in germinating homozygous barley ( Hordeum vulgare L.) grain with thioredoxin h overexpressed in starchy endosperm. Results were related to the null segregant in which the transgene had segregated out during crossing. Compared with the null segregant, the homozygote showed enhanced germination and root and shoot growth in the presence of 1 and 2 mM sodium selenite. The rate of incorporation of selenite by the homozygote was approximately twice that of the null segregant. Based on X-ray absorption spectroscopy, the major products in both cases were selenomethionine-like species and the red, monoclinic form of elemental selenium, a derivative not previously reported in green plants. Selenite and selenate made up the balance. The distribution of the products formed differed as to the tissue - root, shoot, aleurone, endosperm - but the ratios were similar in the homozygote and null segregant. The results provide evidence that, in addition to the accelerated germination observed previously in water, barley grain overexpressing thioredoxin h are resistant to the inhibitory effects of selenite. These properties raise the possibility that plants overexpressing thioredoxin h could find application in the remediation of polluted environments.

Unraveling thioredoxin-linked metabolic processes of cereal starchy endosperm using proteomics.

Application of a thiol-specific probe, monobromobimane, with proteomics and enzyme assays led to the identification of 23 thioredoxin targets in the starchy endosperm of mature wheat seeds (Triticum aestivum cv. Butte), almost all containing at least two conserved cysteines. The identified targets, 12 not known to be thioredoxin-linked, function in a spectrum of processes: metabolism (12 targets), protein storage (three), oxidative stress (three), protein degradation (two), protein assembly/folding (one) and unknown reactions (two). In addition to formulating metabolic pathways functional in the endosperm, the results suggest that thioredoxin acts in redox regulation throughout the life cycle of the seed.

Thioredoxin and germinating barley: targets and protein redox changes.

The endosperm and embryo of barley ( Hordeum vulgare L.) grain were investigated to relate thioredoxin h and disulfide changes to germination and seedling development. The disulfide proteins of both tissues were found to undergo reduction following imbibition. Reduction reached a peak 1 day earlier in the embryo than in the endosperm, day 1 vs. day 2. The profile in both cases resembled those observed with wheat and rice, i.e., the reduction of the storage proteins increased initially and then declined during the period of seedling growth. The extent of the increase in reduction observed with barley endosperm was, however, less pronounced than with the other cereals. Also, unlike wheat and rice, the storage proteins of the endosperm were highly reduced in the dry seed and the sulfhydryl content of glutelins showed no appreciable change during this period. The relative abundance of thioredoxin h during germination and early seedling growth differed in the embryo and endosperm: a progressive decrease in the endosperm (as seen with wheat) vs. an increase in the embryo. Thioredoxin h was found in the major seed tissues in characteristic forms. Three forms were found in the scutellum and aleurone, whereas two, which may represent isoforms, were identified in the root and the shoot. Using a recently developed strategy based on two-dimensional gel electrophoresis, several proteins were identified as specific targets for thioredoxin in the embryo following oxidation with H(2)O(2), among them barley embryo globulin 1, peroxiredoxin and acidic ribosomal protein P(3). The results confirm earlier findings with the endosperm of other cereals and extend the importance of thioredoxin-linked redox change to the germinating embryo for functions that potentially include dormancy, protection against reactive oxygen species, translation and the mobilization of storage proteins.

Transgenic barley grain overexpressing thioredoxin shows evidence that the starchy endosperm communicates with the embryo and the aleurone.

Homozygous lines of barley overexpressing a wheat thioredoxin h transgene (up to 30-fold) were generated earlier by using a B(1)-hordein promoter with a signal peptide sequence for targeting to the protein body and found to be enriched in starch debranching enzyme (pullulanase). Here, we describe the effect of biochemically active, overexpressed thioredoxin h on germination and the onset of alpha-amylase activity. Relative to null segregant controls lacking the transgene, homozygotes overexpressing thioredoxin h effected (i) an acceleration in the rate of germination and appearance of alpha-amylase activity with a 1.6- to 2.8-fold increase in gibberellin A(1) (GA(1)) content; (ii) a similar acceleration in the appearance of the alpha-amylase activity in deembryonated transgenic grain incubated with gibberellic acid; (iii) a 35% increase in the ratio of relative reduction (abundance of SH) of the propanol soluble proteins (hordein I fraction); and (iv) an increase in extractable and soluble protein of 5-12% and 11-35%, respectively. Thioredoxin h, which was highly reduced in the dry grain, was degraded in both the null segregant and homozygote after imbibition. The increase in alpha-amylase activity and protein reduction status was accompanied by a shift in the distribution of protein from the insoluble to the soluble fraction. The results provide evidence that thioredoxin h of the starchy endosperm communicates with adjoining tissues, thereby regulating their activities, notably by accelerating germination of the embryo and the appearance of alpha-amylase released by the aleurone.