Multi-omic analysis of the effects of low frequency ventilation during cardiopulmonary bypass surgery.

BACKGROUND: Heart surgery with cardio-pulmonary bypass (CPB) is associated with lung ischemia leading to injury and inflammation. It has been suggested this is a result of the lungs being kept deflated throughout the duration of CPB. Low frequency ventilation (LFV) during CPB has been proposed to reduce lung dysfunction. METHODS: We used a semi-biased multi-omic approach to analyse lung biopsies taken before and after CPB from 37 patients undergoing coronary artery bypass surgery randomised to both lungs left collapsed or using LFV for the duration of CPB. We also examined inflammatory and oxidative stress markers from blood samples from the same patients. RESULTS: 30 genes were induced when the lungs were left collapsed and 80 by LFV. Post-surgery 26 genes were significantly higher in the LFV vs. lungs left collapsed, including genes associated with inflammation (e.g. IL6 and IL8) and hypoxia/ischemia (e.g. HIF1A, IER3 and FOS). Relatively few changes in protein levels were detected, perhaps reflecting the early time point or the importance of post-translational modifications. However, pathway analysis of proteomic data indicated that LFV was associated with increased "cellular component morphogenesis" and a decrease in "blood circulation". Lipidomic analysis did not identify any lipids significantly altered by either intervention. DISCUSSION: Taken together these data indicate the keeping both lungs collapsed during CPB significantly induces lung damage, oxidative stress and inflammation. LFV during CPB increases these deleterious effects, potentially through prolonged surgery time, further decreasing blood flow to the lungs and enhancing hypoxia/ischemia.

The cardiac proteome in patients with congenital ventricular septal defect: A comparative study between right atria and right ventricles.

Right ventricle (RV) remodelling occurs in neonatal patients born with ventricular septal defect (VSD). The presence of a defect between the two ventricles allows for shunting of blood from the left to right side. The resulting RV hypertrophy leads to molecular remodelling which has thus far been largely investigated using right atrial (RA) tissue. In this study we used proteomic and phosphoproteomic analysis in order to determine any difference between the proteomes for RA and RV. Samples were therefore taken from the RA and RV of five infants (0.34 ±â€¯0.05 years, mean ±â€¯SEM) with VSD who were undergoing cardiac surgery to repair the defect. Significant differences in protein expression between RV and RA were seen. 150 protein accession numbers were identified which were significantly lower in the atria, whereas none were significantly higher in the atria compared to the ventricle. 19 phosphorylation sites (representing 19 phosphoproteins) were also lower in RA. This work has identified differences in the proteome between RA and RV which reflect differences in contractile activity and metabolism. As such, caution should be used when drawing conclusions based on analysis of the RA and extrapolating to the hypertrophied RV. SIGNIFICANCE: RV hypertrophy occurs in neonatal patients born with VSD. Very little is known about how the atria responds to RV hypertrophy, especially at the protein level. Access to tissue from age-matched groups of patients is very rare, and we are in the unique position of being able to get tissue from both the atria and ventricle during reparative surgery of these infants. Our findings will be beneficial to future research into heart chamber malformations in congenital heart defects.

Senescent cancer-associated fibroblasts secrete active MMP-2 that promotes keratinocyte dis-cohesion and invasion.

BACKGROUND: Previous studies have demonstrated that senescent cancer-associated fibroblasts (CAFs) derived from genetically unstable oral squamous cell carcinomas (GU-OSCC), unlike non-senescent CAFs from genetically stable carcinomas (GS-OSCC), promoted keratinocyte invasion in vitro in a paracrine manner. The mechanism by which this occurs is unclear. METHODS: Previous work to characterise the senescent-associated secretory phenotype (SASP) has used antibody arrays, technology that is limited by the availability of suitable antibodies. To extend this work in an unbiased manner, we used 2D gel electrophoresis and mass spectroscopy for protein identification. Matrix metalloproteinases (MMPs) were investigated by gelatin zymography and western blotting. Neutralising antibodies were used to block key molecules in the functional assays of keratinocyte adhesion and invasion. RESULTS: Among a variety of proteins that were differentially expressed between CAFs from GU-OSCC and GS-OSCC, MMP-2 was a major constituent of senescent CAF-CM derived from GU-OSCC. The presence of active MMP-2 was confirmed by gelatine zymography. MMP-2 derived from senescent CAF-CM induced keratinocyte dis-cohesion and epithelial invasion into collagen gels in a TGF-ß-dependent manner. CONCLUSIONS: Senescent CAFs from GU-OSCC promote a more aggressive oral cancer phenotype by production of active MMP-2, disruption of epithelial adhesion and induction of keratinocyte invasion.

Identification and isolation of cDNA clones encoding the abundant secreted proteins in the saliva proteome of Culicoides nubeculosus.

Culicoides spp. are vectors of several infectious diseases of veterinary importance and a major cause of allergy in horses and other livestock. Their saliva contains a number of proteins which enable blood feeding, enhance disease transmission and act as allergens. We report the construction of a novel cDNA library from Culicoides nubeculosus linked to the analysis of abundant salivary gland proteins by mass spectrometry. Fifty-four novel proteins sequences are described including those of the enzymes maltase, hyaluronidase and two serine proteases demonstrated to be present in Culicoides salivary glands, as well as several members of the D7 family and protease inhibitors with putative anticoagulant activity. In addition, several families of abundant proteins with unknown function were identified including some of the major candidate allergens that cause insect bite hypersensitivity in horses.

Identification of abundant proteins and potential allergens in Culicoides nubeculosus salivary glands.

IgE-mediated type 1 hypersensitivity reactions to the bites of insects are a common cause of skin disease in horses. Insect bite hypersensitivity (IBH) is most frequently associated with bites of Culicoides spp. and occurs in all parts of the world where horses and Culicoides coexist. The main allergens that cause IBH are probably some of the abundant proteins in the saliva of Culicoides associated with blood feeding. Western blots of Culicoides proteins separated by 1D gel-electrophoresis detected strong IgE responses in all horses with IBH to antigens in protein extracts from wild caught Culicoides, but only weak responses to salivary antigens from captive bred C. nubeculosus which may reflect important differences among allergens from different species of Culicoides or differences between thorax and salivary gland antigens. 2D electrophoresis and mass spectrometry were used to identify several of the abundant proteins in the saliva of C. nubeculosus. These included maltase, members of the D7 family, and several small, basic proteins associated with blood feeding. The most frequently detected IgE-binding proteins were in a group of proteins with pI>8.5 and mass 40-50kDa. Mass spectrometry identified two of these allergenic proteins as similar to hyaluronidase and a heavily glycosylated protein of unknown function that have previously been identified in salivary glands of C. sonorensis.

14-3-3 proteins within the hypothalamic-neurohypophyseal system of the osmotically stressed rat: transcriptomic and proteomic studies.

The hypothalamic-neurohypophyseal system (HNS) mediates neuroendocrine responses to dehydration through the actions of the antidiuretic hormone vasopressin (VP) and the natriuetic peptide oxytocin (OT). VP and OT are synthesised as separate prepropeptide precursors in the cell bodies of magnocellular neurones in the hypothalamic supraoptic nucleus (SON) and paraventricular nucleus, the axons of which innervate the posterior pituitary gland (PP). Dehydration evokes a massive release of both peptides into the circulation, and this is accompanied by a function-related remodelling of the HNS. Microarray studies on mRNAs differentially expressed in the SON revealed that transcripts encoding the Ywhag and Ywhaz isoforms of the 14-3-3 family of regulatory proteins, are increased in the rat SON by 3 days of water deprivation; findings that we have confirmed by the real-time polymerase chain reaction. Because there is no necessary proportionality between transcript and protein abundance, we next examined Ywhag and Ywhaz translation products throughout the HNS in parallel with 14-3-3 post-translational modification, which is known to be an important determinant of functional activity. Both proteins are robustly expressed in the SON in VP- and OT-containing neurones, but the abundance of neither changes with dehydration. However, the total level of Ywhaz protein is increased in the neurointermediate lobe of the pituitary (NIL, which includes the PP), in parallel with a basic post-translationally modified isoform, suggesting transport from the cell bodies of the SON of newly-synthesised protein and changes in its activity. The level of an acidic, probably phosphorylated, Ywhag isoform is down-regulated in the SON by dehydration, although total levels are unchanged. Finally, based on the presence of a phosphorylated 14-3-3 binding motif, we have identified a 14-3-3 binding partner, proteasome subunit, beta type 7, in the NIL. Thus, we suggest that, through complex transcriptional, and post-translational processes, 14-3-3 proteins are involved in the regulation or mediation of HNS plasticity following dehydration.

Dehydration-induced proteome changes in the rat hypothalamo-neurohypophyseal system.

The hypothalamo-neurohypophyseal system (HNS) mediates neuroendocrine responses to dehydration through the action of the antidiuretic hormone vasopressin (VP). VP is synthesized as part of a prepropeptide in magnocellular neurons of the hypothalamic supraoptic nucleus (SON) and paraventricular nucleus. This precursor is processed during transport to axon terminals in the posterior pituitary gland, in which biologically active VP is stored until mobilized for secretion by electrical activity evoked by osmotic cues. During release, VP travels through the blood stream to specific receptor targets located in the kidney in which it increases the permeability of the collecting ducts to water, reducing the renal excretion of water, thus promoting water conservation. The HNS undergoes a dramatic function-related plasticity during dehydration. We hypothesize that alterations in steady-state protein levels might be partially responsible for this remodeling. We investigated dehydration-induced changes in the SON and pituitary neurointermediate lobe (NIL) proteomes using two-dimensional fluorescence difference gel electrophoresis. Seventy proteins were altered by dehydration, including 45 in the NIL and 25 in the SON. Using matrix-assisted laser desorption/ionization mass spectrometry, we identified six proteins in the NIL (four down, two up) and nine proteins in the SON (four up, five down) that are regulated as a consequence of chronic dehydration. Results for five of these proteins, namely Hsp1alpha (heat shock protein 1alpha), NAP22 (neuronal axonal membrane protein 22), GRP58 (58 kDa glucose regulated protein), calretinin, and ProSAAS (proprotein convertase subtilisin/kexin type 1 inhibitor), have been confirmed using independent methods such as semiquantitative Western blotting, two-dimensional Western blotting, enzyme-linked immunoassay, and immunohistochemistry. These proteins may have roles in regulating and effecting HNS remodeling.

Cell cycle-dependent phosphorylation of the translational repressor eIF-4E binding protein-1 (4E-BP1).

A fundamental control point in the regulation of the initiation of protein synthesis is the formation of the eukaryotic initiation factor 4F (eIF-4F) complex. The formation of this complex depends upon the availability of the mRNA cap binding protein, eIF-4E, which is sequestered away from the translational machinery by the tight association of eIF-4E binding proteins (4E-BPs). Phosphorylation of 4E-BP1 is critical in causing its dissociation from eIF-4E, leaving 4E available to form translationally active eIF-4F complexes, switching on mRNA translation. In this report, we provide the first evidence that the phosphorylation of 4E-BP1 increases during mitosis and identify Ser-65 and Thr-70 as phosphorylated sites. Phosphorylation of Thr-70 has been implicated in the regulation of 4E-BP1 function, but the kinase phosphorylating this site was unknown. We show that the cyclin-dependent kinase, cdc2, phosphorylates 4E-BP1 at Thr-70 and that phosphorylation of this site is permissive for Ser-65 phosphorylation. Crucially, the increased phosphorylation of 4E-BP1 during mitosis results in its complete dissociation from eIF-4E.

Dissociation of the eukaryotic initiation factor-4E/4E-BP1 complex involves phosphorylation of 4E-BP1 by an mTOR-associated kinase.

mTOR immunoprecipitates contain two 4E-BP1 protein kinase activities. One appears to be due to mTOR itself and results in the phosphorylation of 4E-BP1 on residues T(36) and T(45), as shown previously by others. The other is a kinase which can be separated from mTOR and which phosphorylates 4E-BP1 within a peptide(s) containing residues S(64) and T(69). This phosphorylation, which occurs predominantly on S(64), results in the dissociation of 4E-BP1 from eIF-4E.

Regulation of protein-synthesis elongation-factor-2 kinase by cAMP in adipocytes.

Treatment of primary rat epididymal adipocytes or 3T3-L1 adipocytes with various agents which increase cAMP led to the phosphorylation of eukaryotic translation elongation factor-2 (eEF-2). The increase in eEF-2 phosphorylation was a consequence of the activation of eEF-2 kinase (eEF-2K), which is a Ca2+/calmodulin-dependent kinase. eEF-2K was shown to be essentially inactive at less than 0.1 microM free Ca2+ when measured in cell-free extracts. Treatment of adipocytes with isoproterenol induced Ca2+-independent eEF-2K activity, and an 8-10-fold activation of eEF-2K was observed at Ca2+ concentrations of less than 0.1 microM. Increased cAMP in 3T3-L1 adipocytes led to the inhibition of total protein synthesis and decreased the rate of polypeptide-chain elongation. We also show that the phosphorylation of eEF-2 and the activity of eEF-2K are insulin-regulated in adipocytes. These results demonstrate a novel mechanism for the control of protein synthesis by hormones which act by increasing cytoplasmic cAMP.

Insulin-stimulated kinase from rat fat cells that phosphorylates initiation factor 4E-binding protein 1 on the rapamycin-insensitive site (serine-111).

The effects of insulin and rapamycin on the phosphorylation of the translation regulator, initiation factor 4E-binding protein 1 (4E-BP1) have been studied in rat fat cells by following changes in the incorporation of 32P from [32P]Pi under steady-state conditions. Both unbound 4E-BP1 and 4E-BP1 bound to eukaryotic initiation factor 4E (eIF4E) were isolated from the cells and then digested with trypsin and other proteases; the radiolabelled phosphopeptides were then separated by two-dimensional thin- layer analysis and HPLC. The results provide confirmation of the conclusion of Fadden, Haystead and Lawrence [J. Biol. Chem. (1997) 272, 10240-10247] that insulin increases the phosphorylation of four sites that fit a Ser/Thr-Pro motif (Thr-36, Thr-45, Ser-64 and Thr-69) and that taken together these phosphorylations result in the dissociation of 4E-BP1 from eIF4E. The effects of insulin on the phosphorylation of these sites, and hence dissociation from eIF4E, are blocked by rapamycin. However, the present study also provides evidence that insulin increases the phosphorylation of 4E-BP1 bound to eIF4E on a further site (Ser-111) and that this is by a rapamycin-insensitive mechanism. Extraction of rat epididymal fat cells followed by chromatography on Mono-S and Superose 12 columns resulted in the separation of both an insulin-stimulated eIF4E kinase and an apparently novel kinase that is highly specific for Ser-111 of 4E-BP1. The 4E-BP1 kinase was activated more than 10-fold by incubation of the cells with insulin and was markedly more active towards 4E-BP1 bound to eIF4E than towards unbound 4E-BP1. The effects of insulin were blocked by wortmannin, but not by rapamycin. A 14-mer peptide based on the sequence surrounding Ser-111 of 4E-BP1 was also a substrate for the kinase, but peptide substrates for other known protein kinases were not. The kinase is quite distinct from casein kinase 2, which also phosphorylates Ser-111 of 4E-BP1. The possible importance of these kinases in the phosphorylation of 4E-BP1 in fat cells is discussed. It is suggested that the phosphorylation of Ser-111 might be a priming event that facilitates the subsequent phosphorylation of Thr-36, Thr-45, Ser-64 and Thr69 by a rapamycin-sensitive process that initiates the dissociation of 4E-BP1 from eIF4E and hence the formation of the eIF4F complex.

Purification and characterisation of an insulin-stimulated protein-serine kinase which phosphorylates acetyl-CoA carboxylase.

An insulin-stimulated protein kinase specific for acetyl-CoA carboxylase has been purified from rat epididymal adipose tissue using Mono-Q chromatography. The kinase binds to (and phosphorylates) the relatively inactive, dimeric form of acetyl-CoA carboxylase, but not to its active, polymeric form, and this property has been used to purify the kinase. Under the conditions used, phosphorylation by the purified kinase did not result in a detectable increase in acetyl-CoA carboxylase activity. These studies also led to the recognition of an 'activator' protein which is capable of increasing the activity of acetyl-CoA carboxylase without changing its phosphorylation state. It is suggested that this 'activator' protein, together with the insulin-activated acetyl-CoA carboxylase kinase, may play a role in the activation of acetyl-CoA carboxylase by insulin.

Multiple signalling pathways involved in the stimulation of fatty acid and glycogen synthesis by insulin in rat epididymal fat cells.

We have investigated the signalling pathways involved in the stimulation of glycogen and fatty acid synthesis by insulin in rat fat cells using wortmannin, an inhibitor of phosphatidylinositol 3-kinase, and rapamycin, which blocks activation of p70 ribosomal S6 protein kinase (p70S6K). Insulin produced a decrease in the activity of glycogen synthase kinase-3 which is likely to be important in the observed stimulation of glycogen synthase. Both of these actions were found to be sensitive to inhibition by wortmannin. Activation of three processes is involved in the stimulation of fatty acid synthesis from glucose by insulin, namely glucose uptake, acetyl-CoA carboxylase and pyruvate dehydrogenase. Whereas wortmannin largely abolished the effects of insulin on glucose utilization and acetyl-CoA carboxylase activity, it was without effect on the stimulation of pyruvate dehydrogenase. Although epidermal growth factor stimulated mitogen-activated protein kinase to a greater extent than insulin, it was unable to mimic the effect of insulin on glycogen synthase, glycogen synthase kinase-3, glucose utilization, acetyl-CoA carboxylase or pyruvate dehydrogenase. Rapamycin also failed to have any appreciable effect on stimulation of these parameters by insulin, although it did block the effect of insulin on p70S6K. We conclude that the activity of phosphatidylinositol 3-kinase is required for the effects of insulin on glycogen synthesis, glucose uptake and acetyl-Co-AN carboxylase, but is not involved in signalling to pyruvate dehydrogenase. Activation of mitogen-activated protein kinase or p70S6K, however, does not appear to be sufficient to bring about the stimulation of fatty acid or glycogen synthesis. Altogether is seems likely that at least four distinct signalling pathways are involved in the effects of insulin on rat fat cells.

Chain-length dependency of interactions of medium-chain fatty acids with glucose metabolism in acini isolated from lactating rat mammary glands. A putative feed-back to control milk lipid synthesis from glucose.

The effects of a series of medium-chain fatty acids (C6-C12) on glucose metabolism in isolated acini from lactating rat mammary glands have been studied. Hexanoate (C6) octanoate (C8) and decanoate (C10), but not laurate (C12), decreased [1-14C]glucose conversion into [14C]lipid and the production of 14CO2 (an index of the pentose phosphate pathway). With hexanoate and octanoate, glucose utilization was decreased, whereas decanoate had a slight stimulatory effect on glucose utilization, but there was a large accumulation of lactate. Addition of dichloroacetate (an inhibitor of pyruvate dehydrogenase kinase) decreased this accumulation of lactate and stimulated the conversion of [1-14C]glucose into [14C]lipid and 14CO2. Insulin had no effect on the rate of glucose utilization in the presence of hexanoate. It stimulated the rate in the presence of octanoate and laurate and increased the conversion of [1-14C]glucose into [14C]lipid in the presence of octanoate, decanoate or laurate. The major fate of 1-14C-labelled medium-chain fatty acids (C6, C8 and C12) was conversion into [14C]lipid. The proportion converted into 14CO2 decreased with increasing chain length, whereas the rate of [14C]lipid formation increased. It is concluded that the interactions between medium-chain fatty acids and glucose metabolism represent a feed-back mechanism to control milk lipid synthesis, and this may be important when milk accumulates in the gland.