Epsilon Protein Research Articles

Chronic Exposure to Light Reverses the Effect of Maternal Separation on Proteins in the Prefrontal Cortex

Animals subjected to maternal separation display behavioural and endocrine disturbances, as well as structural and functional changes in the prefrontal cortex and limbic areas. The aim of the present study was to determine the effect of maternal separation and treatment with either chronic constant light exposure or anti-depressant (escitalopram) on proteins in the prefrontal cortex. Four experimental groups of male Sprague-Dawley rats were subjected to (1) normal rearing, (2) maternal separation (3 h per day from postnatal day 2 (P2) to P14), (3) maternal separation followed by chronic light exposure (P42-P63) or (4) maternal separation followed by treatment with the anti-depressant drug, escitalopram (P68-P100). Groups 1-3 were treated with saline as vehicle control for the escitalopram-treated group. At P101, all rats were decapitated, and the prefrontal cortex was collected and stored at -80 °C. Tissue from three rats per group was pooled and proteins determined by isobaric tagging for relative and absolute quantification using matrix-assisted laser desorption/ionisation tandem mass spectrometry. Maternal separation led to disruptions in the prefrontal cortex that included hypometabolism by decreasing energy-related proteins (creatine kinase B, aconitate hydratase), decreased cell signalling (synapsin I, calmodulin, 14-3-3 protein epsilon) and impaired plasticity (spectrin, microtubule-associated protein). Maternal separation also increased dihydropyrimidinase-related protein/collapsin response mediator protein (CRMP) and myelin proteolipid protein. Exposure of maternally separated animals to constant light during adolescence reversed the hypometabolic state by increasing energy-related proteins in the prefrontal cortex and increasing cell signalling and cytoskeletal proteins and decreasing the expression of CRMP. Escitalopram had similar effects to light by increasing ATP synthase in maternally separated rats and dissimilar effects by increasing 2',3'-cyclic-nucleotide 3'-phosphodiesterase and myelin proteolipid protein. Constant light exposure during adolescence reversed a range of protein changes in the prefrontal cortex of rats exposed to early maternal separation. The most prominent reversal by light treatment of maternal separation-induced protein increases in the prefrontal cortex was the expression of CRMP which impairs plasticity and neuronal signalling. The effects of light treatment overlapped partially with the effects of escitalopram.

Identification of functional nucleotide and haplotype variants in the promoter of the CEBPE gene

The current study examined the promoter activity of an association signal in a 5'-upstream region of the gene encoding CCAAT/enhancer binding protein epsilon (CEBPE) identified from a recent genome-wide association study (GWAS) for complex acute lymphoblastic leukemia (ALL). This follow-up study first compared the activity of reporter constructs with three haplotypes estimated with the rs2239633 and its proximity nucleotide variants in strong linkage. The most frequent haplotype was CTTTTGT (H1), and the second most frequent haplotype consisted of entirely opposite alleles to H1 (TCGCACC, H2). Their luciferase activity revealed the strongest expression with H2 and the weakest with H1. Subsequent analysis revealed that different luciferase activity was found by the single-nucleotide substitution at rs2239632 and rs2239633 (P<0.05). Especially, the difference in luciferase activity between two alleles of rs2239632 corresponded to the difference between H1 and H2. We concluded that rs2239632 could regulate the expression of the CEBPE gene. We suggest a hypothesis that its risk allele (G) might increase the gene product and lead to leukemogenesis. As a result, a person with the allele or the corresponding haplotype might have increased susceptibility to ALL. Further research is warranted to investigate this hypothesis and the underlying mechanisms.

Abstract 5448: Genome-wide ChIP-seq of C/EBPε reveals networks controlling myelopoiesis .

Abstract CAAT/enhancer-binding protein epsilon (C/EBPε) is known to be a critical transcription factor mediating terminal differentiation of granulocytes and a target to boost killing of bacteria by the innate immune system. However, our data found that ∼9 month old C/EBPε knockout (KO) mice developed a novel phenotype similar to the myeloproliferative disorder. In bone marrow (BM) of C/EBPε KO mice, colonies of granulocyte-macrophage progenitor (CFU-GM) were increased (143±21 vs. 59±13, P&amp;lt;0.01) while erythroid progenitors (BFU-E) were decreased (8±2 vs.14±4, P&amp;lt;0.01) as compared to age- and sex- matched wild type controls. Flow data showed that the percentage of BM immature myeloid (Mac1+Gr1int) were much higher (48%±3% vs. 9%±5%, P&amp;lt;0.01) while Ter119+ erythrocytes were less (13%±4% vs. 47%±1%, P&amp;lt;0.01) than control. In addition, the ratio of spleen/ body weight in C/EBPε KO mice was augmented (0.81%±0.16% vs. 0.25%±0.03%, P&amp;lt;0.01) and spleen H&amp;E staining showed extramedullary hemopoiesis. Compared to spleen samples in control mice, CFU-GM (97±16 vs. 9±2, P&amp;lt;0.01), BFU-E (20±5 vs. 4±1, P&amp;lt;0.01) and multi-potential granulocyte, erythroid, macrophage, megakaryocyte progenitors (CFU-GEMM, 11±3 vs. 2±1, P&amp;lt;0.01) in C/EBPε KO mice were significantly increased. Consistent with spleen CFU assay, the percentage of Mac1+Gr1int immature myeloid cells (18%±8% vs. 3%±1%, P&amp;lt;0.05) and Ter119+ erythrocytes (38%±1% vs. 12%±2%, P&amp;lt;0.01) were raised in KO than in control mice by flow cytometry analysis. Taken together, C/EBPε deletion in mice blocks myeloid differentiation and impairs erythoid development in BM as well as leads to extramedullary hemopoiesis in spleen. To investigate regulatory networks controlled by C/EBPε, we performed genome-wide chromatin immunoprecipitation followed by sequencing (ChIP-seq) in wild type and C/EBPε KO mice BM using N- and C-terminal C/EBPε antibodies. We have identified ∼15000 significant C/EBPε binding sites and as with the majority of transcription factors, C/EBPε targets predominantly distal intergenic and intronic regulatory regions. However, 15% of significant binding sites are localized within gene promoters and some of them are known to play a critical role in hematopoietic lineage differentiation such as PU.1, RUNX1 and SCL. Interestingly, C/EBPε also binds known distal regulatory regions as the PU.1 enhancer, and our data has highlighted novel potential intergenic and intronic enhancers for RUNX1. The ChIP-PCR results of these transcription factors are consistent with ChIP-seq. In summary, our study suggests that C/EBPε can have a profound effect on myeloid development by regulating key genes and its deregulation can produce a myeloproliferative abnormality. Citation Format: Tong Yin, Touati Benoukraf, Phillip H. Koeffler. Genome-wide ChIP-seq of C/EBPε reveals networks controlling myelopoiesis . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5448. doi:10.1158/1538-7445.AM2013-5448 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

The Safety and Efficacy of KAI-1678— An Inhibitor of Epsilon Protein Kinase C (εPKC)—Versus Lidocaine and Placebo for the Treatment of Postherpetic Neuralgia: A Crossover Study Design

Postherpetic neuralgia (PHN) occurs in approximately 10-20% of patients with herpes zoster, and the risk increases with age. In this clinical trial, we evaluated the analgesic properties of KAI-1678-an inhibitor of epsilon protein kinase C-in the treatment of neuropathic pain in patients with PHN. The study was a three-treatment period, double-blind, randomized, placebo and active comparator crossover trial evaluating subcutaneous infusions of KAI-1678 (25 mg), placebo, and lidocaine hydrochloride (700 mg; active comparator). A total of 17 men and 6 women (N = 23) were enrolled after fulfilling diagnosis of PHN with pain persisting for ≥3 months after a segmental herpes zoster eruption. Patients had to have a mean average pain score of ≥4 points on an 11-point numerical rating scale (NRS; ranging from 0 to 10) based on at least three daily entries prior to participation in the subsequent treatment period. Overall, administration of KAI-1678 was generally safe and well tolerated. However, compared with placebo, KAI-1678 did not improve clinical pain scores as recorded using the NRS (0-10). In contrast, subcutaneous infusions of lidocaine were associated with a significant reduction in pain intensity at the end of the infusion. We conclude that KAI-1678 is not efficacious as an acute analgesic for chronic neuropathic pain because of PHN. However, for the first time, the results demonstrate that subcutaneous infusions of lidocaine are effective in treating neuropathic pain. The results of lidocaine treatment also indicate that the crossover study design was adequate to detect a clinically meaningful response in this analgesia study.

The transcription factor activating enhancer‐binding protein epsilon (AP–2ε) regulates the core promoter of type II collagen (COL2A1)

The transcription factor activating enhancer-binding protein epsilon (AP-2ε) was recently shown to be expressed during late chondrocyte differentiation, especially in hypertrophic chondrocytes. In this study, we were able to reveal that the promoter of the type II collagen (COL2A1) gene, encoding the extracellular matrix protein type II collagen, is specifically regulated by AP-2ε. Expression of COL2A1 is downregulated at the transition of chondroblasts into hypertrophic chondrocytes and our data provide evidence that AP-2ε is involved in this process. In reporter gene assays, overexpression of AP-2ε in cartilaginous cell lines resulted in a significant reduction in COL2A1 core promoter activity of ~ 45%. Furthermore, we found that this process is dose-dependent and highly specific for the epsilon isoform. Computational analysis offered only a single putative AP-2-binding motif within the chosen promoter fragment but site-directed mutagenesis revealed this motif to be regulatory inactive. After expanding our screening to motifs containing minor differences from the classical AP-2 consensus sequence (5'-GCCN3 GGC-3'), we determined the sequence 5'-GCCCAGGC-3' ranging from position -128 to -135 bp as an important regulatory site and responsible for COL2A1 downregulation through AP-2ε. Interaction of AP-2ε with the COL2A1 promoter at this site was confirmed by chromatin immunoprecipitation and electromobility shift assay. Further, our experiments suggest that at least one additional factor is involved in this process. This is the first study to prove regulation of COL2A1 by AP-2ε highlighting the role of the transcription factor as a modulator of cartilage development.

Interaction of Ubinuclein-1, a nuclear and adhesion junction protein, with the 14-3-3 epsilon protein in epithelial cells: Implication of the PKA pathway

Ubinuclein-1 is a NACos (Nuclear and Adhesion junction Complex components) protein which shuttles between the nucleus and tight junctions, but its function in the latter is not understood. Here, by co-immunoprecipitation and confocal analysis, we show that Ubinuclein-1 interacts with the 14-3-3ɛ protein both in HT29 colon cells, and AGS gastric cells. This interaction is mediated by an Ubinuclein-1 phosphoserine motif. We show that the arginine residues (R56, R60 and R132) which form the 14-3-3ɛ ligand binding site are responsible for the binding of 14-3-3ɛ to phosphorylated Ubinuclein-1. Furthermore, we demonstrate that in vitro Ubinuclein-1 can be directly phosphorylated by cAMP-dependent protein kinase A. This in vitro phosphorylation allows binding of wildtype 14-3-3ɛ. Moreover, treatment of the cells with inhibitors of the cAMP-dependent protein kinase, KT5720 or H89, modifies the subcellular localization of Ubinuclein-1. Indeed, KT5720 and H89 greatly increase the staining of Ubinuclein-1 at the tight junctions in AGS gastric cells. In the presence of the kinase inhibitor KT5720, the amount of Ubinuclein-1 in the NP40 insoluble fraction is increased, together with actin. Moreover, treatment of the cells with KT5720 or H89 induces the concentration of Ubinuclein-1 at tricellular intersections of MDCK cells. Taken together, our findings demonstrate novel cell signaling trafficking by Ubinuclein-1 via association with 14-3-3ɛ following Ubinuclein-1 phosphorylation by the cAMP-dependent protein kinase-A.

MiR-1 Exacerbates Cardiac Ischemia-Reperfusion Injury in Mouse Models

Recent studies have revealed the critical role of microRNAs (miRNAs) in regulating cardiac injury. Among them, the cardiac enriched microRNA-1(miR-1) has been extensively investigated and proven to be detrimental to cardiac myocytes. However, solid in vivo evidence for the role of miR-1 in cardiac injury is still missing and the potential therapeutic advantages of systemic knockdown of miR-1 expression remained unexplored. In this study, miR-1 transgenic (miR-1 Tg) mice and locked nucleic acid modified oligonucleotide against miR-1 (LNA-antimiR-1) were used to explore the effects of miR-1 on cardiac ischemia/reperfusion injury (30 min ischemia followed by 24 h reperfusion). The cardiac miR-1 level was significantly increased in miR-1 Tg mice, and suppressed in LNA-antimiR-1 treated mice. When subjected to ischemia/reperfusion injury, miR-1 overexpression exacerbated cardiac injury, manifested by increased LDH, CK levels, caspase-3 expression, apoptosis and cardiac infarct area. On the contrary, LNA-antimiR-1 treatment significantly attenuated cardiac ischemia/reperfusion injury. The expression of PKCε and HSP60 was significantly repressed by miR-1 and enhanced by miR-1 knockdown, which may be a molecular mechanism for the role miR-1 in cardiac injury. Moreover, luciferase assay confirmed the direct regulation of miR-1 on protein kinase C epsilon (PKCε) and heat shock protein 60 (HSP60). In summary, this study demonstrated that miR-1 is a causal factor for cardiac injury and systemic LNA-antimiR-1 therapy is effective in ameliorating the problem.

Antibacterial Activity of Selected Standard Strains of Lactic Acid Bacteria Producing Bacteriocins – Pilot Study

In this paper, an attempt was made to evaluate the antibacterial potential of standard strains of lactic acid bacteria (LAB) producing bacteriocins of various classes, thus demonstrating various mechanisms of cell membrane damages against the Streptococcus agalactiae strains (Group B Streptococcus, GBS), depending on surface polysaccharides and surface alpha-like protein genes. Antimicrobial property of the strains of L. plantarum C 11, L. sakei DSMZ 6333, and L. lactis ATCC 11454 producing bacteriocins: JK and EF plantaricins, sakacin and nisin, respectively, against the GBS strains was evaluated. The chosen to the study GBS strains were represented by serotypes Ia, Ib, II, III, V and they had bca, epsilon, rib, alp2 or alp3 alpha-like protein genes. The experiment was conducted by means of suspension culture and the bacteria count was determined using the serial dilution method. A great ability of L. plantarum C 11 strain was proven to inhibit the GBS growth. The strain of L. sakei DSMZ 6333 did not demonstrate any ability to inhibit the growth of GBS, whereas L. lactis ATCC 11454 inhibited the growth of S. agalactiae indicator strains to a minor extent. Statistically significant differences were demonstrated between the GBS strains representing various serotypes against the antimicrobial activity of model LAB strains. The least sensitive to the activity of bacteriocins were the strains representing serotypes Ib and III, whereas the strains representing serotype II were the most sensitive. The sensitivity of the GBS strains to the antimicrobial activity of LAB was not dependent on alpha-like protein genes. Among the LAB standard strains producing bacteriocins, the strongest antimicrobial property was observed in the strain of L. plantarum C 11. Because of the generally known and verified strong antagonistic property of the strains of L. plantarum species against indicator bacteria, it is necessary to further pursue the research presented in this paper.

Genetic polymorphisms in ARID5B,CEBPE,IKZF1 and CDKN2A in relation with risk of acute lymphoblastic leukaemia in adults: a Group for Research on Adult Acute Lymphoblastic Leukaemia (GRAALL) study

Genetic polymorphisms in <i><scp>ARID</scp>5B</i>,<i><scp>CEBPE</scp></i>,<i><scp>IKZF</scp>1</i> and <i><scp>CDKN</scp>2A</i> in relation with risk of acute lymphoblastic leukaemia in adults: a <scp>G</scp>roup for <scp>R</scp>esearch on <scp>A</scp>dult <scp>A</scp>cute <scp>L</scp>ymphoblastic <scp>L</scp>eukaemia (GRAALL) study

Formation of nitrated tryptophan residues as a novel post-translational modification in the physiological state

Protein nitration is a post-translational modification that is induced by reactive nitrogen species (RNS) such as peroxynitrite (ONOO−) and nitrogen dioxide (NO2). Nitration of tyrosine residues in proteins has been studied extensively and found in many diseases under oxidative stress. We have found a novel type of protein nitration, formation of 6-nitrotryptophan (6-NO2Trp) residues, developed its specific antibody, and constructed a method to detect 6-NO2Trp-containing proteins by proteomic analysis using LC–ESI–MS–MS (1). In this study, we applied this method for PC12 cell (naïve PC12 cell) and NGF-treated PC12 cell (differentiated-PC12 cell) to detect 6-NO2Trp-containing proteins in physiological state. We found several peptides from five ribosomal proteins (RP) (60S RP L7, 60S acidic RP P1, 40s RP S2, 40S RP S6, and 40S RP S19), 14-3-3 protein epsilon, nucleoside diphosphate kinase B, and proteasome subunit alpha type1 as nitrotryptophan containing peptides. We successfully determined the positions of nitrated tryptophan residues in the amino acid sequences of these proteins. Among them, the tryptophan nitration was observed only in the differentiated-PC12 cells for 40S RP S19 protein and 14-3-3 protein. The nitrotryptophan positive signals of 40S RP S19 and 14-3-3 protein were suppressed by treatment of PC12 cells with NOS inhibitor, L-NAME. The tryptophan nitration was not observed by LC–MS–MS analysis of these proteins. Finally, we also found several tryptophan-nitrated proteins in brain of adult rats by using the same method. Reference [1] H. Kawasaki et al., Free Radic. Biol. Med. 50 (2011) 419–427.

Abstract 2412: Epithelial splicing regulatory protein 1 modulates cell growth, migration, and invasion of human pancreatic ductal adenocarcinoma

Abstract Background: Recent studies have shown that epithelial splicing regulatory protein 1 (ESRP1) plays important roles in the epithelial mesenchymal transition of cancer cells via regulating the splicing of several RNAs, including fibroblast growth factor receptor 2 (FGFR2). Previously we reported that the expression of FGFR2IIIb correlates with vascular invasion and liver metastasis of pancreatic ductal adenocarcinoma (PDAC) (Cho, et al. Am J Pathol. 2008), while the expression of FGFR2IIIc correlates with the growth and invasion of PDAC cells (Ishiwata, et al. AACR. 2010). However, the expression and roles of ESRP1 in PDAC have not been well-defined. Methods: We performed immunohistochemical analysis of ESRP1, FGFR2IIIb, and FGFR2IIIc using pancreatic tissue specimens of PDAC (N=52) and non-tumorous pancreatic tissue (N=8). The ESRP1 expression vector and small interference RNA (siRNA) targeting ESRP1 were used to transfect human PDAC cells, and cell growth, morphology, migration, and invasion were analyzed. To clarify the ESRP1-regulating molecules, we performed proteomic analysis of ESRP1-gene-transfected PDAC cells using mass spectrometry. Results: In immunohistochemical analysis, ESRP1 was strongly expressed in the nuclei of PDAC cells in well- and moderately differentiated adenocarcinoma, while weakly expressed in poorly differentiated adenocarcinoma and normal pancreatic ducts. Well-moderately differentiated adenocarcinomas showed high-FGFR2IIIb/low-FGFR2IIIc pattern, with opposite for poorly differentiated adenocarcinoma. The overall and disease-free survival rates of the high-ESRP1 group were significantly than those of the low-ESRP1 group. In vitro, transient transfection of PDAC cells with ESRP1 expression vector led to an increase in FGFR2IIIb expression and decrease in migration and invasion. Stable transfection of PDAC cells with the ESRP1 expression vector resulted in an increase of FGFR2IIIb expression and decrease in migration and invasion. In contrast, ESRP1 siRNA-transfected PDAC cells induced FGFR2IIIc expression and increased cell growth, migration, and invasion. In proteomic analysis, transiently ESRP1 transfected PDAC cells showed a decreased expression of vimentin, 14-3-3 protein epsilon, heat shock protein 70, and the IQ motif containing GTPase activating protein 1, but an increased expression of filamin alpha. Several proteins as determined by proteomic analysis are involved in cell growth, migration, and invasion. Conclusion: The expression of ESRP1 correlates with the differentiation and prognosis of PDAC, and it modulates cell growth, migration, and invasion in PDAC cells via the alternation of several proteins including FGFR2. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2412. doi:1538-7445.AM2012-2412

Sustained inhibition of epsilon protein kinase C inhibits vascular restenosis after balloon injury and stenting

Sustained inhibition of epsilon protein kinase C inhibits vascular restenosis after balloon injury and stenting

Activation of Epsilon Protein Kinase C-Mediated Anti-Apoptosis Is Involved in Rapid Tolerance Induced by Electroacupuncture Pretreatment Through Cannabinoid Receptor Type 1

Our previous study has demonstrated that the rapid tolerance to cerebral ischemia by electroacupuncture (EA) pretreatment was possibly mediated through an endocannabinoid system-related mechanism. The purpose of this study was to investigate whether activation of epsilon protein kinase C (εPKC) was involved in EA pretreatment-induced neuroprotection via cannabinoid receptor type 1 in a rat model of transient focal cerebral ischemia. The activation of εPKC in the ipsilateral brain tissues after EA pretreatment was investigated in the presence or absence of cannabinoid receptor antagonists. At 2 hours after the end of EA pretreatment, focal cerebral ischemia was induced by middle cerebral artery occlusion for 120 minutes in rats. The neurobehavioral scores, infarction volumes, neuronal apoptosis, and the expression of Bcl-2 and Bax were evaluated after reperfusion in the presence or absence of εPKC-selective peptide inhibitor (TAT-εV1-2) or activator (TAT-ψεRACK). EA pretreatment enhanced εPKC activation. Systemic delivery of TAT-ψεRACK conferred neuroprotection against a subsequent cerebral ischemic event when delivered 2 hours before ischemia. Pretreatment with EA reduced infarct volumes, improved neurological outcome, inhibited neuronal apoptosis, and increased the Bcl-2-to-Bax ratio after reperfusion, and the beneficial effects were attenuated by TAT-εV1-2. In addition, the blockade of cannabinoid receptor type 1, but not cannabinoid receptor type 2 receptor, reversed the increase in εPKC activation and neuroprotection induced by EA pretreatment. EA pretreatment may activate endogenous εPKC-mediated anti-apoptosis to protect against ischemic damage after focal cerebral ischemia via cannabinoid receptor type 1, which represents a new mechanism of EA pretreatment-induced rapid tolerance to focal cerebral ischemia in rats.

CCAAT/Enhancer Binding Protein Epsilon Mediates Nicotinamide-Enhanced Clearance of Staphylococcus Aureus Infection

AbstractAbstract 925Staphylococcus aureus in community and healthcare settings commonly causes serious and potentially life-threatening infections. Widespread use of antibiotics is responsible for the emergence and rapid spread of resistant pathogens, including methicillin-resistant S. aureus (MRSA), and highlights a pressing need for development of novel antimicrobial therapies. The myeloid-specific transcription factor, CCAAT/enhancer binding protein epsilon (C/EBPε) serves as a critical regulator of the terminal differentiation and functional maturation of neutrophils and macrophages, both crucial components of the innate immune system.Comparable to humans with neutrophil specific granule deficiency (SGD) carrying a causative mutation in this transcription factor, we showed that C/EBPε-deficient (C/EBPε—/—) mice were severely affected by in vivo infection with S. aureus. Paradoxically, depletion of the defective neutrophils attenuated disease pathology and even improved the outcome of infection. During subcutaneous infection with S. aureus, C/EBPε—/— mice treated with mouse anti-polymorphonuclear neutrophil antibody showed significantly smaller skin lesions, fewer CFU within the lesion, and reduced systemic spread of bacteria. In addition, whole blood from C/EBPε—/— mice was less effective at killing S. aureus compared to their cell-free plasma. Therefore, ineffective clearance of S. aureus by C/EBPε—/— neutrophils, even compared to extracellular killing mechanisms, likely permitted S. aureus to thrive within neutrophils, which further aggravated the infection.Because C/EBPε plays a critical role in the host immune response against S. aureus infection, we further hypothesized that increased activity of C/EBPε could enhance immune killing of bacteria. Using a zink-inducible expression vector, we induced overexpression of C/EBPε in U937-macrophages, and thereby enhanced bacterial clearance including MRSA by up to 1.5 log10 CFU/mL. Interestingly, we found that the epigenetic modulator, nicotinamide (NAM; vitamin B3), increased activity of C/EBPε as well as downstream antimicrobial targets. Upon exposing bone-marrow derived macrophages or mononuclear cells from wildtype mice to NAM (1 or 10 mM), increased levels of lysine acetylation on core histone H3 were detected at the promoter region of CEBPE. This was associated with elevated mRNA and protein levels of C/EBPε, and increased expression of downstream antimicrobials such as cathelicidin(-related) antimicrobial peptide (CAMP) and Lactoferrin. In an in vitro, as well as in vivo infection model, moderately concentrated NAM enhanced killing of S. aureus by up to 3 log10, but had no effect when administered to C/EBPε-deficient mice. This again points to C/EBPε as an important target to boost killing of bacteria by the innate immune system. Strikingly, and consistent with our murine data, NAM treatment reduced the ability of S. aureus to survive in whole human blood obtained from 12 healthy humans by 2–3 log10. In line with our findings on S. aureus, we were able to demonstrate similar immune boosting effects of NAM in human blood infected with other important human pathogens such as K. pneumoniae and P. aeruginosa.In an age when the number of antibiotics in the pipeline is limited and development of resistance occurs rapidly, use of complementary strategies to antibiotic treatment provides a promising method of limiting development of antibiotic resistance. Here, we demonstrated that C/EBPε is a regulatory factor that critically impacts the host's ability to fight bacterial infections. Compounds exerting modulatory effects on this myeloid-specific transcription factor may emerge as important antimicrobial therapeutics against frequent pathogens such as S. aureus. Disclosures:No relevant conflicts of interest to declare.

Differential Protein Expression in Honeybee (Apis mellifera L.) Larvae: Underlying Caste Differentiation

Honeybee (Apis mellifera) exhibits divisions in both morphology and reproduction. The queen is larger in size and fully developed sexually, while the worker bees are smaller in size and nearly infertile. To better understand the specific time and underlying molecular mechanisms of caste differentiation, the proteomic profiles of larvae intended to grow into queen and worker castes were compared at 72 and 120 hours using two dimensional electrophoresis (2-DE), network, enrichment and quantitative PCR analysis. There were significant differences in protein expression between the two larvae castes at 72 and 120 hours, suggesting the queen and the worker larvae have already decided their fate before 72 hours. Specifically, at 72 hours, queen intended larvae over-expressed transketolase, aldehyde reductase, and enolase proteins which are involved in carbohydrate metabolism and energy production, imaginal disc growth factor 4 which is a developmental related protein, long-chain-fatty-acid CoA ligase and proteasome subunit alpha type 5 which metabolize fatty and amino acids, while worker intended larvae over-expressed ATP synthase beta subunit, aldehyde dehydrogenase, thioredoxin peroxidase 1 and peroxiredoxin 2540, lethal (2) 37 and 14-3-3 protein epsilon, fatty acid binding protein, and translational controlled tumor protein. This differential protein expression between the two caste intended larvae was more pronounced at 120 hours, with particular significant differences in proteins associated with carbohydrate metabolism and energy production. Functional enrichment analysis suggests that carbohydrate metabolism and energy production and anti-oxidation proteins play major roles in the formation of caste divergence. The constructed network and validated gene expression identified target proteins for further functional study. This new finding is in contrast to the existing notion that 72 hour old larvae has bipotential and can develop into either queen or worker based on epigenetics and can help us to gain new insight into the time of departure as well as caste trajectory influencing elements at the molecular level.

Proteomic analysis of differentially expressed proteins in Penaeus monodon hemocytes after Vibrio harveyi infection

BackgroundViral and bacterial diseases can cause mass mortalities in commercial shrimp aquaculture. In contrast to studies on the antiviral response, the responses of shrimps to bacterial infections by high throughput techniques have been reported only at the transcriptional level and not at the translational level. In this study, a proteomic analysis of shrimp hemocytes to identify differentially expressed proteins in response to a luminous bacterium Vibrio harveyi was evaluated for its feasibility and is reported for the first time.ResultsThe two-dimensional gel electrophoresis (2-DE) patterns of the hemocyte proteins from the unchallenged and V. harveyi challenged shrimp, Penaeus monodon, at 24 and 48 h post infection were compared. From this, 27 differentially expressed protein spots, and a further 12 weakly to non-differentially regulated control spots, were selected for further analyses by the LC-ESI-MS/MS. The 21 differentially expressed proteins that could be identified by homologous annotation were comprised of proteins that are directly involved in the host defense responses, such as hemocyanin, prophenoloxidase, serine proteinase-like protein, heat shock protein 90 and alpha-2-macroglobulin, and those involved in signal transduction, such as the14-3-3 protein epsilon and calmodulin. Western blot analysis confirmed the up-regulation of hemocyanin expression upon bacterial infection. The expression of the selected proteins which were the representatives of the down-regulated proteins (the 14-3-3 protein epsilon and alpha-2-macroglobulin) and of the up-regulated proteins (hemocyanin) was further assessed at the transcription level using real-time RT-PCR.ConclusionsThis work suggests the usefulness of a proteomic approach to the study of shrimp immunity and revealed hemocyte proteins whose expression were up regulated upon V. harveyi infection such as hemocyanin, arginine kinase and down regulated such as alpha-2-macroglobulin, calmodulin and 14-3-3 protein epsilon. The information is useful for understanding the immune system of shrimp against pathogenic bacteria.

Cardioprotection by Regular Ethanol Consumption: Potential Mechanisms and Clinical Application

Epidemiological studies demonstrate that excessive drinking is associated with hypertension, cerebral bleeding and loss of cardiac contractility. Conversely, studies have shown that mortality rates for people who regularly drink ethanol in moderation are lower than in abstainers, primarily due to decreased fatal ischemic heart disease. Further, moderate ethanol consumers have lower rates of myocardial infarction compared with abstainers. These beneficial cardiac effects may be due to pleiotropic effects of ethanol on lipids, platelets, and fibrinolytic activity. During the past decade, studies conducted in several animal models have revealed that light to moderate regular ethanol consumption renders hearts more tolerant to myocardial ischemia-reperfusion injury; to a degree similar to cardiac ischemic preconditioning (brief episodes of ischemia dramatically limit infarct size following prolonged ischemia). Recent clinical evidence suggests that light to moderate ethanol consumption in the year prior to myocardial infarction is associated with reduced mortality following myocardial infarction. These findings suggest that light to moderate ethanol consumption not only prevents myocardial infarction but also improves survival after myocardial infarction. Proposed mechanisms of cardioprotection by regular ethanol consumption include activation of adenosine A1 receptors, alpha(1)-adrenoceptors, protein kinase C-delta and epsilon, adenosine triphosphate-dependent potassium (K(ATP)) channels, nitric oxide synthase and reduced leukocyte-endothelial cell adhesive interactions. In this review, we focus on the recent progress in elucidating the endogenous myocyte signaling mediating cardioprotection by light to moderate ethanol consumption.

Differential Protein Levels and Post-Translational Modifications in Spinal Cord Injury of the Rat

Although changes in protein expression in spinal cord injury (SCI) would be of pivotal interest, information so far is limited. It was therefore the aim of the study to determine protein levels and post-translational modifications in the early phase following SCI in the rat. SCI was induced in Sprague-Dawley rats and sham operated rats served as controls. A gel-based proteomic approach using two-dimensional gel electrophoresis followed by quantification with specific software and subsequent identification of differentially expressed proteins by nano-ESI-LC-MS/MS was applied. Proteins of several pathways and cascades were dysregulated in SCI: 14-3-3 epsilon protein, dynein light chain 1, and tubulin beta-5 chain showed higher levels in SCI, whereas adenylyl cyclase associated protein 1, dihydropyrimidinase-related protein 2, F-actin capping protein subunit beta, glyceraldehyde-3-phosphate dehydrogenase, stress-induced phosphoprotein 1 and transthyretin showed lower levels in the injured tissue. Post-translational modifications indicated free oxygen radical attack on proteins in SCI. The occurrence of stress is indicated by deranged stress-induced phosphoprotein 1 and signaling abnormalities are reflected by adenylyl cyclase-associated protein 1 and 14-3-3 epsilon protein. The findings propose the involvement of the corresponding cascades and challenge further work into aberrant signaling and oxidative stress in SCI, which may form the basis for experimental intervention for spinal cord trauma.

Ischemic preconditioning mediates cyclooxygenase-2 expression via nuclear factor-kappa B activation in mixed cortical neuronal cultures.

Nuclear factor-kappaB (NF-κB) activation occurs following ischemic preconditioning (IPC) in brain. However, the upstream signaling messengers and down-stream targets of NF-κB required for induction of IPC remain undefined. In a previous study, we demonstrated that epsilon protein kinase c (εPKC) was a key mediator of IPC in brain. Activation of εPKC induced cyclooygenase-2 (COX-2) expression and conferred ischemic tolerance in the neuronal and hippocampal slice models. Here, we hypothesized that IPC-mediated COX-2 expression was mediated by NF-κB. We tested this hypothesis in mixed cortical neuron/astrocyte cell cultures. To simulate IPC or ischemia, cell cultures were exposed to 1 or 4 h of oxygen-glucose deprivation, respectively. Our results demonstrated translocation of p65 and p50 subunits of NF-κB into nucleus following IPC or εPKC activation. NF-κB inhibition with pyrrolidine dithiocarbamate (10 μM) abolished IPC or εPKC activator-mediated neuroprotection indicating that NF-κB activation was involved in ischemic tolerance. In parallel studies, inhibition of either εPKC or the extracellular signal-regulated kinase (ERK 1/2) pathway reduced IPC-induced NF-κB activation. Finally, inhibition of NF-κB blocked IPC-induced COX-2 expression. In conclusion, we demonstrated that IPC-signaling cascade comprises εPKC activation→ERK1/2 activation→NF-κB translocation to nucleus→COX-2 expression resulting in neuroprotection in mixed neuronal culture.

Identifying PKC epsilon as a target molecule to control intimal hyperplasia

Background: Epsilon protein kinase C (εPKC) is a PKC isoform that plays pivotal roles in myocardial infarction and in heart failure. Here we investigate whether PKC epsilon activation is also involved in the development of intimal hyperplasia.