Phosphatidylethanolamine-binding Protein Research Articles

Hepatocellular Protein Profiles After Hepatic Ischemia/Reperfusion Injury With or Without Octreotide Preconditioning in a Rabbit Model

Hepatic ischemic/reperfusion injury (HIRI) is a major complication of liver resection and transplantation. Octreotide, a somatostatin analogue, has been used to treat hepatic fibrosis and portal hypertension; however, its function against HIRI remains unclear. To elucidate the effect of octreotide in HIRI, we investigated the hepatocellular protein profiles in response to octreotide preconditioning in a rabbit model by using proteomic analysis. Twenty-four rabbits were divided into 3 groups: the sham operative group (control), the ischemia/reperfusion group (IR), and the ischemia/reperfusion + octreotide group (IR+Oct). They were subjected to 30 minutes of normothermic ischemia followed by 120 minutes of reperfusion by using Pringle's maneuver method. Proteomic studies were then performed to compare the protein profiles of their left liver lobe. A total of 16 differential proteins were successfully identified. These findings suggest that octreotide might exert an effect against HIRI through up-regulating the expression of the anti-injury substances, such as heat-shock proteins 70 and 27 (confirmed by using Western blot analysis); significantly raising the phosphatidylethanolamine-binding protein that alleviates IR-related apoptosis; and down-regulating mitochondrial metabolic enzymes such as NADH2 dehydrogenase and triosephosphate isomerase.

The floral transcriptome of Eucalyptus grandis.

As a step toward functional annotation of genes required for floral initiation and development within the Eucalyptus genome, we used short read sequencing to analyze transcriptomes of floral buds from early and late developmental stages, and compared these with transcriptomes of diverse vegetative tissues, including leaves, roots, and stems. A subset of 4807 genes (13% of protein-coding genes) were differentially expressed between floral buds of either stage and vegetative tissues. A similar proportion of genes were differentially expressed among all tissues. A total of 479 genes were differentially expressed between early and late stages of floral development. Gene function enrichment identified 158 gene ontology classes that were overrepresented in floral tissues, including 'pollen development' and 'aromatic compound biosynthetic process'. At least 40 floral-dominant genes lacked functional annotations and thus may be novel floral transcripts. We analyzed several genes and gene families in depth, including 49 putative biomarkers of floral development, the MADS-box transcription factors, 'S-domain'-receptor-like kinases, and selected gene family members with phosphatidylethanolamine-binding protein domains. Expanded MADS-box gene subfamilies in Eucalyptus grandis included SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1), SEPALLATA (SEP) and SHORT VEGETATIVE PHASE (SVP) Arabidopsis thaliana homologs. These data provide a rich resource for functional and evolutionary analysis of genes controlling eucalypt floral development, and new tools for breeding and biotechnology.

Downregulation of PEBP4, a target of miR-34a, sensitizes drug-resistant lung cancer cells.

The aim of this study was to determine the relationship and underlying mechanisms between ectopic expression of phosphatidylethanolamine-binding protein 4 (PEBP4) and cisplatin (DDP)-induced cytotoxicity in the lung cancer cell line A549 to provide an experimental basis for future chemotherapeutic applications involving PEBP4 in human lung cancer. A recombinant plasmid, pcDNA3-PEBP4, and a PEBP4-targeting small hairpin RNA (shRNA) were transfected into the lung cancer cell line A549. The PEBP4 protein expression levels were determined for each group by Western blot, and after 48 h of cisplatin (DDP) treatment, the viability of cells in the treatment and control groups was determined by 3-[4,5-dimethylthiazol-2-yl]-3,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis in each treatment group was determined using flow cytometry. Western blotting was used to examine expression of the p53 protein in A549 cells from each group. We employed a luciferase reporter-gene assay to confirm PEBP4 as a target gene of miR-34a. Western blotting was used to determine the effects of miR-34a on PEBP4 protein expression in A549 cells. Following transfection of A549 cells with either the recombinant plasmid pcDNA3-PEBP4 or a PEBP4-targeting shRNA, Western blotting analyses showed PEBP4 protein expression was significantly higher in the pcDNA3-PEBP4-transfected group compared with the control or PEBP4-shRNA-transfected groups (p < 0.01). Furthermore, PEBP4 protein expression was significantly reduced in the PEBP4-shRNA-transfected group (p < 0.01). After 48 h of DDP treatment, MTT assays indicated that A549 cell viability was significantly lower in the DDP-treated group compared with the control group (p < 0.01). The viability of A549 cells in the pcDNA3-PEBP4-transfected group was lower than that in the control group (p < 0.05) but higher than that in either the DDP-treated or PEBP4-shRNA-transfected groups (p < 0.05). Moreover, the viability of A549 cells in the PEBP4-shRNA-transfected group was significantly lower than that in either the control (p < 0.01) or DDP-treated (p < 0.05) groups. Flow cytometry and Western blotting analyses indicated that the number of apoptotic cells and p53 protein expression were significantly higher in the DDP-treated group compared with the control group (p < 0.01). In the pcDNA3-PEBP4-transfected group, the number of apoptotic cells and p53 protein expression level were higher than those in the control group (p < 0.05) but lower than those in the DDP-treated and PEBP4-shRNA-transfected groups (p < 0.05). The number of apoptotic cells and p53 protein expression level in the PEBP4-shRNA-transfected group were higher than those in the control (p < 0.01) and DDP-treated (p < 0.05) groups. The luciferase reporter-gene assay showed that the relative luciferase activity after transfection with a miR-34a mimic was significantly reduced compared with the control group (p < 0.01). Western blotting analysis demonstrated that PEBP4 protein expression was significantly decreased in A549 cells 48 h after transfection with a miR-34a mimic compared with the control group (p < 0.01). In conclusion, overexpression of PEBP4 reduced the sensitivity of A549 cells to DDP-induced cytotoxicity, mainly through the altered expression of the p53 protein or the modulation of miR-34a.

Comprehensive identification of essential pathways and transcription factors related to epilepsy by gene set enrichment analysis on microarray datasets.

Epilepsy is a common chronic neurological disorder characterized by seizures or convulsions, and is known to affect patients with primary brain tumors. The etiology of epilepsy is superficially thought to be multifactorial; however, the genetic factors which may be involved in the pathogenesis of seizures have not yet been elucidated, particularly at the pathway level. In the present study, in order to systematically investigate the gene regulatory networks involved in epilepsy, we employed a microarray dataset from the public database library of Gene Expression Omnibus (GEO) associated with tumor-induced epileptogenesis and applied gene set enrichment analysis (GSEA) on these data sets and performed candidate transcription factor (TF) selection. As a result, 68 upregulated pathways, including the extracellular matrix (ECM)-receptor interaction (P=0.004) and peroxisome proliferator-activated receptor (PPAR) signaling pathways (P=0.045), as well as 4 downregulated pathways, including the GnRH signaling pathway (P=0.029) and gap junction (P=0.034) were identified as epileptogenesis-related pathways. The majority of these pathways identified have been previously reported and our results were in accordance with those reports. However, some of these pathways identified were novel. Finally, co-expression networks of the related pathways were constructed with the significant core genes and TFs, such as PPAR-γ and phosphatidylethanolamine-binding protein. The results of our study may contribute to the improved understanding of the molecular mechanisms of epileptogenesis on a genome-wide level.

Differential proteomic profile of spermatogenic and Sertoli cells from peri-pubertal testes of three different bovine breeds.

Sub-fertility is one of the most common problems observed in crossbred males, but the etiology remains unknown in most of the cases. Although proteomic differences in the spermatozoa and seminal plasma between breeds have been investigated, the possible differences at the sperm precursor cells and supporting/nourishing cells have not been studied. The present study reports the differential proteomic profile of spermatogenic and Sertoli cells in crossbred and purebred bulls. Testis was removed by unilateral castration of 12 peri-pubertal bulls (10 months age), four each from crossbred (Holstein Friesian × Tharparkar), exotic purebred [Holstein Friesian (HF)] and indigenous purebred [Tharparkar (TP)] bulls. Spermatogenic and Sertoli cells were isolated and subjected to proteomic analysis. Protein extracts from the Sertoli and spermatogenic cells of each breed were analyzed with 2-dimensional difference gel electrophoresis (2D-DIGE) and analyzed with Decyder™ software. Compared to HF, 26 protein spots were over expressed and 14 protein spots were under expressed in spermatogenic cells of crossbred bulls. Similarly, 7 protein spots were over expressed and 15 protein spots were under expressed in the spermatogenic cells of TP bulls compared to that of crossbred bulls. Out of 12 selected protein spots identified through mass spectrometry, Phosphatidyl ethanolamine binding protein was found to be over expressed in the spermatogenic cells of crossbred bulls compared to TP bulls. The protein, gamma actin was found to be over expressed in the Sertoli cells of HF bulls, whereas Speedy Protein-A was found to be over expressed in Sertoli cells of crossbred bulls. It may be concluded that certain proteomic level differences exist in sperm precursor cells and nourishing cells between breeds, which might be associated with differences in the fertility among these breeds.

Putative sugarcane FT/TFL1 genes delay flowering time and alter reproductive architecture in Arabidopsis.

Agriculturally important grasses such as rice, maize, and sugarcane are evolutionarily distant from Arabidopsis, yet some components of the floral induction process are highly conserved. Flowering in sugarcane is an important factor that negatively affects cane yield and reduces sugar/ethanol production from this important perennial bioenergy crop. Comparative studies have facilitated the identification and characterization of putative orthologs of key flowering time genes in sugarcane, a complex polyploid plant whose genome has yet to be sequenced completely. Using this approach we identified phosphatidylethanolamine-binding protein (PEBP) gene family members in sugarcane that are similar to the archetypical FT and TFL1 genes of Arabidopsis that play an essential role in controlling the transition from vegetative to reproductive growth. Expression analysis of ScTFL1, which falls into the TFL1-clade of floral repressors, showed transcripts in developing leaves surrounding the shoot apex but not at the apex itself. ScFT1 was detected in immature leaves and apical regions of vegetatively growing plants and, after the floral transition, expression also occurred in mature leaves. Ectopic over-expression of ScTFL1 in Arabidopsis caused delayed flowering in Arabidopsis, as might be expected for a gene related to TFL1. In addition, lines with the latest flowering phenotype exhibited aerial rosette formation. Unexpectedly, over-expression of ScFT1, which has greatest similarity to the florigen-encoding FT, also caused a delay in flowering. This preliminary analysis of divergent sugarcane FT and TFL1 gene family members from Saccharum spp. suggests that their expression patterns and roles in the floral transition has diverged from the predicted role of similar PEBP family members.

Molecular mechanism of Raf kinase inhibitory protein regulation by cAMP‐dependent protein kinase (802.25)

Raf Kinase Inhibitory Protein (RKIP), a 21 kDa protein and member of phosphatidylethanolamine binding protein (PEBP) family, regulates GPCR, MAPK, and NFκB pathways and is a metastasis suppressor. Although, kinases such as ERK2 and PKCα have been shown to phosphorylate RKIP, structural data on the protein kinase‐RKIP interaction are still lacking. Here we report that another kinase, PKA, phosphorylates RKIP at residue 51, triggering a feedback loop, and we determine the structural model of RKIP‐PKA catalytic subunit. Using different biophysical approaches including Nuclear Magnetic Resonance (NMR), Paramagnetic Relaxation Enhancements (PREs) and molecular modeling, we identify the binding interface between RKIP and PKA catalytic subunit. Chemical shift mapping and site directed mutagenesis studies revealed residues within RKIP that are critical to the catalysis and binding mediated by PKA catalytic subunit. As the first description of PKA binding to a full‐length physiological substrate, these results have wide applicability to the mechanism of PKA catalysisGrant Funding Source: Supported by NIH grant GM087630 to Marsha Rich Rosner, NIH grant GM100310 to Gianluigi Veglia

Structural Characterization of a Phospho‐Switch in the Metastasis Suppressor RKIP (LB189)

RKIP is a member of the phosphatidylethanolamine binding protein (PEBP) family with over 400 members. Previous work showed that phosphorylation of S153 was sufficient to alter RKIP binding partners, effectively upregulating the MAP kinase and β‐adrenergic receptor signaling pathways (Granovsky and Rosner, Cell Res., 18:452, 2008). Yet little is known regarding the mechanism by which RKIP phosphorylation regulates these key signaling pathways. Here we propose a three state allosteric model to explain RKIP function. Using NMR, we demonstrate that the RKIP phospho‐switch is associated with a major structural change that can be instigated by breaking a single salt bridge. We also demonstrate that RKIP can dimerize as a result of the phospho‐switch due to formation of disulfide linkages and the functional significance of this oligomerization will be presented. Together these results reveal a novel phospho‐switch that regulates RKIP function and has broad applicability to other PEBP family members throughout evolution.

Raf Kinase Inhibitor Protein (RKIP) Blocks Signal Transducer and Activator of Transcription 3 (STAT3) Activation in Breast and Prostate Cancer

Raf kinase inhibitor protein (RKIP) is a member of the phosphatidylethanolamine-binding-protein (PEBP) family that modulates the action of many kinases involved in cellular growth, apoptosis, epithelial to mesenchymal transition, motility, invasion and metastasis. Previously, we described an inverse association between RKIP and signal transducers and activators of transcription 3 (STAT3) expression in gastric adenocarcinoma patients. In this study, we elucidated the mechanism by which RKIP regulates STAT3 activity in breast and prostate cancer cell lines. RKIP over expression inhibited c-Src auto-phosphorylation and activation, as well as IL-6-, JAK1 and 2-, and activated Raf-mediated STAT3 tyrosine and serine phosphorylation and subsequent activation. In MDA-231 breast cancer cells that stably over express RKIP, IL-6 treatment blocked STAT3 phosphorylation and transcriptional activation. Conversely, in RKIP knockdown MDA-231 cells: STAT3 phosphorylation and activation increased in comparison to parental MDA-231 cells. RKIP over expression resulted in constitutive physical interaction with STAT3 and blocked c-Src and STAT3 association. The treatment of DU145 prostate, but not PC3 prostate or MDA-231 breast, cancer cell lines with ENMD-1198 or MKC-1 dramatically increased expression of RKIP. Overexpression of RKIP sensitized PC3 and MDA-231 cells to MTI-induced apoptosis. Moreover, MTI treatment resulted in a decrease in Src-mediated STAT3 tyrosine phosphorylation and activation, an effect that was significantly enhanced by RKIP over expression. In stable RKIP over expressing MDA-231 cells, tumor xenograft growth induced by activated STAT3 is inhibited. RKIP synergizes with MTIs to induce apoptosis and inhibit STAT3 activation of breast and prostate cancer cells. RKIP plays a critical role in opposing the effects of pro-oncogenic STAT3 activation.

Human Phosphatidylethanolamine-Binding Protein 4 Promoted the Radioresistance of Human Rectal Cancer by Activating Akt in an ROS-Dependent Way

Human phosphatidylethanolamine-binding protein 4(hPEBP4) is a novel anti-apoptosis molecule associated with the resistance of tumors to apoptotic agents. Here we sought to investigate the role of hPEBP4 in the radioresistance of rectal cancer. Immunohistochemistry analysis showed hPEBP4 was expressed in 27/33 of rectal cancer specimens, but only in 2/33 of neighboring normal mucosa. Silencing the expression of hPEBP4 with siRNA significantly reduced the clonogenic survival and enhanced the apoptosis of rectal cancer cells on irradiation. Instead, forced overexpression of hPEBP4 promoted its survival and decreased the apoptosis. Western blot showed hPEBP4 could increase the radiation-induced Akt activation, for which reactive oxygen specimen(ROS) was required. The radioresistance effect of hPEBP4 was reversed after given LY-294002 to inhibit Akt activation or antioxidant to abolish the ROS production. We also confirmed that effect of hPEBP4 in vivo with nude mice. Thus we concluded that hPEBP4, specifically expressed in rectal cancer cells, is associated with radioresistance of rectal cancer, implying that modulation of hPEBP4 may have important therapeutic implications in radiotherapy of rectal cancer.

Raf kinase inhibitory protein (RKIP): functional pleiotropy in the mammalian brain.

In 1984, a cytosolic protein was isolated from bovine brain and coined phosphatidylethanolamine binding protein (PEBP) to describe its phospholipid-binding potential. Its cellular function remained elusive for more than a decade until it was discovered that PEBP had the ability to suppress the Raf1-mitogen activated protein kinase (MAPK) pathway, earning it the new name of Raf1 kinase inhibitory protein (RKIP). This milestone discovery has paved the way for numerous studies that have now extended the reach of RKIP's function to other signaling cascades, within the context of various physiological and pathophysiological systems. This review will summarize our current knowledge of the neurophysiological roles of RKIP in the mammalian brain, including its function in the circadian clock and synaptic plasticity. It will also discuss evidence for an involvement of RKIP and its derived neuropeptide, hippocampal cholinergic neurostimulating peptide (HCNP), in neural development and differentiation. Implications in certain pathologies such as Alzheimer's disease and brain cancer will be highlighted. By chronicling the diverse functions of RKIP in the brain, we hope that this review will serve as a timely resource that ignites future studies on this versatile, multifaceted protein in the nervous system.

Molecular characterization and functional analysis of a Flowering locus T homolog gene from a Phalaenopsis orchid.

Warm day and cool night conditions significantly induce reproductive spike formation in Phalaenopsis plants; hence, determining the flowering mechanism regulating the reproductive transition is important. Flowering locus T (FT) plays important roles in flowering induction in several plants. To explore spike induction by warm days and cool nights in Phalaenopsis orchids, we isolated the FT (PhFT) from Phalaenopsis hybrid Fortune Saltzman. The cDNA of PhFT was 809-bp long and contained a 531-bp open reading frame encoding a putative protein of 176 amino acids, a 58-bp 5'-untranslated region (UTR), and a 220-bp 3'-UTR. The predicted molecular mass of PhFT was 19.80 kDa, with an isoelectric point of 8.68. The PhFT was predicted to possess the conserved functional regions of the phosphatidylethanolamine-binding protein superfamily. Nucleotide sequence data indicated that PhFT contained 3 introns and 4 exons. Sequence alignment and phylogenetic analyses of PhFT revealed high homology to the FT proteins of Cymbidium goeringii and Oncidium Gower Ramsey. Quantitative real-time polymerase chain reaction analysis indicated that PhFT mRNA was expressed in roots, apical leaves, mature leaves, and flowers. In flowers, PhFT was expressed more in developing floral buds than in mature flowers and was predominantly expressed in ovaries and petals. Ectopic expression of PhFT in Arabidopsis ft-1 mutants showed novel early-flowering phenotypes that lost their siliques. Our results indicated that the ectopic expression of PhFT could partially complement the late flowering defect in transgenic Arabidopsis ft-1 mutants. Our findings suggest that PhFT is a putative FT homolog in Phalaenopsis plants that regulates flowering transition.

Cigarette Smoking Exposure Alters Pebp1 DNA Methylation and Protein Profile Involved in MAPK Signaling Pathway in Mice Testis1

Many studies have addressed the role of cigarette smoking on semen quality, but the exact mechanisms remain inconclusive. To evaluate the detrimental effects of smoking on the spermatogenesis process, we initially screened and investigated 31 differentially expressed proteins extracted from the testes of mice exposed daily to cigarette smoke using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry analysis. Data mining analysis showed that these 31 proteins were categorized into five functional clustering groups: metabolic process, cell growth and/or maintenance, RNA and protein processing, stress response, and spermatogenesis. Additionally, 23 of 31 proteins were involved in a main pathway network, including Pkc (s), ERK1/2, Akt, and NF-kappaB, which are known to be involved in cell communication, proliferation, and differentiation. Interestingly, among the 31 proteins, a spermatogenesis-associated protein, phosphatidylethanolamine-binding protein 1 (PEBP1), was especially expressed in serial sections of spermatids of spermiogenesis and interacted with ERKs. The bisulfite sequencing result showed four CpGs near the Pebp1 transcriptional start site were largely methylated in the treated group. A 5-aza-2'-deoxycytidine treatment on GC-1 spg cells reversed the hypermethylation status and elevated both Pebp1 mRNA and protein expression levels. ERK1/2 phosphorylation levels were also increased with upregulation of Pebp1 expression in GC-1 spg cells. In conclusion, protein profile in testes could be altered by cigarette smoking. Moreover, we also suggest that epigenetic Pebp1 inactivation may affect activation of ERK, and it could impair spermatogenesis of mice. Our data could provide further insight into the mechanisms of spermatogenesis.

Phosphatidylethanolamine-binding protein 4 is associated with breast cancer metastasis through Src-mediated Akt tyrosine phosphorylation

Metastasis is responsible for more than 90% of the mortality observed among patients with breast cancer. Human phosphatidylethanolamine-binding protein 4 (hPEBP4) is a novel member of the PEBP family and functions as an anti-apoptotic molecule. Here, we found that the metastatic MDA-MB-231 breast cancer cells expressed much higher levels of hPEBP4 than the nonmetastatic MCF-7 breast cancer cells and that the expression levels of hPEBP4 were positively correlated with the metastasis of clinical breast cancer. The hPEBP4 overexpression in the MDA-MB-231 cells significantly promoted cell invasion in vitro and increased the development of lymph node metastasis in vivo. Conversely, the silencing of hPEBP4 suppressed the cell-invasive ability both in vitro and in vivo. Further investigation showed that hPEBP4 promoted the expression or activity of the metastasis-related proteinases MMP (matrix metalloproteinase) 2, MMP9 and MMP13. This hPEBP4-potentiated cell invasion and MMP expression is due to an increase in Akt activation. Knockdown of Akt restored hPEBP4-induced breast tumor metastasis in the hPEBP4-MDA-MB-231 xenograft mouse model. Moreover, we found that hPEBP4 functioned as a scaffolding molecule and enhanced the association of Akt with Src to promote Akt tyrosine phosphorylation, a prerequisite for the full activation of Akt, in a phosphatidylethanolamine-binding domain-dependent manner. Given the present information about human breast cancer, these functional data from cell culture and animal studies suggest that, in human breast cancer hPEBP4 is a novel and clinically relevant metastasis accelerator gene and may be a new diagnostic marker and therapeutic target for breast cancer metastasis.

Regulation of Polyunsaturated Fatty Acid Biosynthesis by Seaweed Fucoxanthin and Its Metabolite in Cultured Hepatocytes

The effects of a seaweed carotenoid, fucoxanthin, and its physiological metabolite, fucoxanthinol, on the biosynthesis of polyunsaturated fatty acids (PUFA) were investigated using cultured rat hepatoma BRL-3A. The metabolism of α-linolenic acid (18:3n-3) was suppressed by the addition of these carotenoids, resulting in a decrease in the content of eicosapentaenoic acid (20:5n-3), which suggested a down-regulation of metabolic enzymes such as fatty acid desaturase and elongase. An increase in the content of docosahexaenoic acid (22:6n-3), as observed in previous studies in vivo, might be a buffering action to maintain the membrane fluidity. The suppressive effect of fucoxanthinol on ∆6 fatty acid desaturase was not at the level of gene expression but due to specific modifications of the protein via a ubiquitin-proteasome system. A proteomic analysis revealed several factors such as phosphatidylethanolamine-binding protein that might be involved in the observed action of fucoxanthin. These findings will contribute to studies on the elucidation of the precise molecular mechanisms underlying the regulation of PUFA biosynthesis by fucoxanthin.

Phosphatidylethanolamine‐Binding Protein 1 (PEBP1) as a Potential Target for the Treatment for Depression

Phosphatidylethanolamine‐Binding Protein 1 (PEBP1) as a Potential Target for the Treatment for Depression

Phosphatidylethanolamine binding protein 1 in vacular endothelial cell autophagy and atherosclerosis

We previously found that phosphatidylcholine-specific phospholipase C (PC-PLC) was a key inducing element of atherosclerosis, and might negatively regulate human umbilical vein endothelial cell (HUVEC) autophagy. To further investigate the mechanism of PC-PLC action, we initially identified phosphatidylethanolamine binding protein 1 (PEBP1) as a binding partner of PC-PLC by using mass spectrometry (MS, MALDI-TOF/TOF). We found that PEBP1 positively regulated PC-PLC activity in HUVECs, and inhibition of PC-PLC by its inhibitor D609 suppressed PEBP1 expression dramatically. Moreover, both PC-PLC and PEBP1 negatively regulated HUVEC autophagy independently of mammalian target of rapamycin (mTOR). Furthermore, the PEBP1 level was elevated during the development of atherosclerosis, while D609 significantly decreased the upregulated PEBP1 level in apoE(-/-) mice.

The utility of isotope-coded protein labeling for prioritization of proteins found in ovarian cancer patient urine

Urine offers a number of attractive features as a sample type for biomarker discovery, including noninvasive sampling, quantity and availability, stability, and a narrow dynamic range. In this study we report the first application of isotope coded protein labeling (ICPL), coupled with in-solution isoelectric fractionation and LC-MALDI-TOF/TOF, to examine and prioritize urinary proteins from ovarian cancer patients. Following the definition of stringent exclusion criteria a total of 579 proteins were identified with 43% providing quantitation data. Protein abundance changes were validated for selected proteins by ESI-Qq-TOF MS, following which Western blot and immunohistochemical analysis by tissue microarray was used to explore the biological relevance of the proteins identified. Several established markers (e.g., HE4, osteopontin) were identified at increased levels in ovarian cancer patient urine, validating the approach used; we also identified a number of potential marker candidates (e.g., phosphatidylethanolamine binding protein 1, cell-adhesion molecule 1) previously unreported in the context of ovarian cancer. We conclude that the ICPL strategy for identification and relative quantitation of urine proteins is an appropriate tool for biomarker discovery studies, and can be applied for the selection of potential biomarker candidates for further characterization.

Differential gene expression in the brain of the African lungfish, Protopterus annectens, after six days or six months of aestivation in air.

The African lungfish, Protopterus annectens, can undergo aestivation during drought. Aestivation has three phases: induction, maintenance and arousal. The objective of this study was to examine the differential gene expression in the brain of P. annectens during the induction (6 days) and maintenance (6 months) phases of aestivation as compared with the freshwater control using suppression subtractive hybridization. During the induction phase of aestivation, the mRNA expression of prolactin (prl) and growth hormone were up-regulated in the brain of P. annectens, which indicate for the first time the possible induction role of these two hormones in aestivation. Also, the up-regulation of mRNA expression of tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein γ polypeptide and the down-regulation of phosphatidylethanolamine binding protein, suggest that there could be a reduction in biological and neuronal activities in the brain. The mRNA expression of cold inducible RNA-binding protein and glucose regulated protein 58 were also up-regulated in the brain, probably to enhance their cytoprotective effects. Furthermore, the down-regulation of prothymosin α expression suggests that there could be a suppression of transcription and cell proliferation in preparation for the maintenance phase. In general, the induction phase appeared to be characterized by reduction in glycolytic capacity and metabolic activity, suppression of protein synthesis and degradation, and an increase in defense against ammonia toxicity. In contrast, there was a down-regulation in the mRNA expression of prl in the brain of P. annectens during the maintenance phase of aestivation. In addition, there could be an increase in oxidative defense capacity, and up-regulation of transcription, translation, and glycolytic capacities in preparation for arousal. Overall, our results signify the importance of reconstruction of protein structures and regulation of energy expenditure during the induction phase, and the needs to suppress protein degradation and conserve metabolic fuel stores during the maintenance phase of aestivation.

Structural and biochemical characterization of Rv2140c, a phosphatidylethanolamine-binding protein from Mycobacterium tuberculosis

Rv2140c is one of many conserved Mycobacterium tuberculosis proteins for which no molecular function has been identified. We have determined a high-resolution crystal structure of the Rv2140c gene product, which reveals a dimeric complex that shares strong structural homology with the phosphatidylethanolamine-binding family of proteins. Rv2140c forms low-millimolar interactions with a selection of soluble phosphatidylethanolamine analogs, indicating that it has a role in lipid metabolism. Furthermore, the small molecule locostatin binds to the Rv2140c ligand-binding site and also inhibits the growth of the model organism Mycobacterium smegmatis.