Peptide 2A Research Articles

Adoption of the 2A Ribosomal Skip Principle to Track Assembled Virions of Pepper Mild Mottle Virus in Nicotiana benthamiana.

The coat protein (CP) is an important structural protein that plays many functional roles during the viral cycle. In this study, the CP of pepper mild mottle virus (PMMoV) was genetically fused to GFP using the foot-and-mouth disease virus peptide 2A linker peptide and the construct (PMMoV-GFP2A) was shown to be infectious. The systemic spread of the virus was monitored by its fluorescence in infected plants. Electron microscopy and immunocolloidal gold labelling confirmed that PMMoV-GFP2A forms rod-shaped particles on which GFP is displayed. Studies of tissue ultrastructure and virion self-assembly confirmed that PMMoV-GFP2A could be used to monitor the real-time dynamic changes of CP location during virus infection. Aggregations of GFP-tagged virions appeared as fluorescent plaques in confocal laser microscopy. Altogether, PMMoV-GFP2A is a useful tool for studying the spatial and temporal changes of PMMoV CP during viral infection.

Synthesized peptide analogs from Eumenes pomiformis (Hymenoptera: Eumenidae) venom reveals their antibiotic and pesticide activity potential

One natural antimicrobial peptide (EpVP2a, Eumenes pomiformis Venom Peptide 2a) found in the venom of a potter wasp (Eumenes pomiformis) and six analogs were synthesized and tested to compare their antimicrobial, antifungal, pesticide, and hemolytic activity with the wild type. Our results indicated that while the original peptide and the synthetic analogs had no antifungal activity or anti-bacterial activity against Pseudomonas aeruginosa, the original peptide and the analog with substitution of the aspartic acid on the sequence by a lysine (EpVP2a-D2K2) had activity against Escherichia coli, Staphylococcus aureus and Bacillus subtilis. This same analog also shows significant insecticide activity. The analog with substitution of lysine with a slightly smaller ornithine had activity against E. coli and B. subtilis. All analogs show low hemolytic activity compared to the natural peptide. The peptide with a reverse sequence to the natural one (EpVp2a Retro) shows low helix structure which can also explain why it has no antibacterial activity and low hemolytic activity. Circular dichroism spectra show that these peptides form an alpha helix structure and their amino acid positions predict an amphipathic nature.

Influence of Dosing Regimen and Adjuvant Type on the Immunogenicity of Novel Recombinant Zika Virus-Like Particles.

Zika virus (ZIKV) is a reemerging mosquito-borne flavivirus that causes febrile illness and is also linked to Guillain-Barré syndrome as well as to microcephaly in newborns. Due to the risk of fetuses developing microcephaly, ZIKV is a serious problem for pregnant women. Although different types of vaccine antigens have been investigated, there is still no approved vaccine that prevents ZIKV. The aim of this study was to produce a potential anti-Zika virus vaccine candidate based on virus-like particles (VLPs) in mammalian cells and to analyze the role of dosing regimen and adjuvant type on the immunogenicity of the obtained antigen. Novel recombinant VLPs (F2A) were designed by introducing the optimized signal sequence of prM protein and by adding a self-cleavage peptide 2A between proteins prM and E. These modifications improved the formation of the glycoprotein E dimer. It has been shown that the increasing dosing regimen generates a significantly higher titer of antibodies; however, the adjuvant type does not affect this process. Sera from mice immunized using an increasing dosing schedule also showed higher neutralization activity against both Zika strains (H/PAN/2016/BEI-259634, a pandemic strain belonging to Asian lineage, and MR766, a reference strain from African lineage). In summary, this is the first report showing the influence of vaccination schedules and adjuvants on the immunogenicity of ZIKV virus-like particles. IMPORTANCE Considering the transmission of ZIKV and the risk of another epidemic as well as the neurological complications that follow ZIKV infection, the virus remains a serious problem for the human population, especially pregnant women. Therefore, there is a great need to develop new effective vaccine candidates. Although different types of vaccine antigens have been used in preclinical studies worldwide, there is still no approved vaccine to prevent ZIKV. VLPs are among the most potent antigens, but to use VLPs, adjuvants must be added to the formulation and appropriate administration must be performed. In this study, we show for the first time the influence of vaccination schedules and adjuvants on the immunogenicity of recombinant ZIKV VLPs. The obtained results can be used in new vaccine designs not only against ZIKV but also against other important viral pathogens.

New Insight on the Immune Modulation and Physical Barrier Protection Caused by Vitamin A in Fish Gills Infected With Flavobacterium columnare

In this study, we have investigated the influence of vitamin A on gill barrier function of grass carp (Ctenopharyngodon idella) infected with Flavobacterium columnare. The fish were fed different concentrations of vitamin A diets for 10 weeks and then infected with F. columnare by immersion. We observed that optimal vitamin A significantly prevented gill rot morbidity in fish infected with F. columnare. Further investigations revealed that vitamin A boosted the gill immunity by increasing the contents of complements (C3 and C4), activities of acid phosphatase (ACP) and lysozyme, mRNAs of β-defensin-1, liver-expressed antimicrobial peptide 2A and 2B (LEAP-2A and LEAP-2B), hepcidin, and anti-inflammatory cytokines like transforming growth factor β1 (TGF-β1), TGF-β2, interleukin-10 (IL-10), and IL-11. It also enhanced the levels of various related signaling molecules including inhibitor protein κBα (IκBα), target of rapamycin (TOR), and ribosome protein S6 kinase 1 (S6K1) but downregulated the expression of pro-inflammatory cytokines including IL-1β, IL-8, tumor necrosis factor α (TNF-α), and interferon γ2 (IFN-γ2) and related signaling molecules including nuclear factor κB p65 (NF-κB p65) (rather than NF-κB p52), IκB kinase β (IKKβ), IKKγ (rather than IKKα), eIF4E-binding protein 1 (4E-BP1), and 4E-BP2 mRNA levels in fish gills. In addition, dietary vitamin A markedly lowered the concentrations of reactive oxygen species (ROS), malondialdehyde (MDA), and protein carbonyl (PC), increased both the activities and mRNAs of copper/zinc superoxide dismutase (Cu/ZnSOD), MnSOD, glutathione transferases (GSTs), glutathione peroxidase (GPx), and glutathione reductase (GR) associated with upregulation of NF-E2-related factor 2 (Nrf2) mRNAs and downregulation of Kelch-like-ECH-associated protein (Keap1a) and Keap1b mRNAs. Moreover, vitamin A decreased the mRNAs of different apoptotic mediators [caspases 8, 9, 3 (rather than 7)] associated with downregulation of signaling molecule p38 mitogen-activated protein kinase (p38MAPK) mRNAs in fish gills. Besides, vitamin A promoted tight junction (TJ) complex mRNAs [including claudin-b, -c, -3, -7, -12, occludin, and zonula occludens-1 (ZO-1)] that have been linked to the downregulation of myosin light chain kinase (MLCK) signaling. Taken together, the current study demonstrated for the first time that vitamin A markedly enhanced gill health associated with immune modulation and physical barrier protection. Based on protecting fish against gill rot morbidity, ACP activity, and against lipid peroxidation, optimum vitamin A concentrations in on-growing grass carp (262–997 g) were found to be 1,991, 2,188, and 2,934 IU/kg diet, respectively.

Identification of vicilin, legumin and antimicrobial peptide 2a as macadamia nut allergens

Macadamia nut is an increasingly popular food item of a healthy diet. However, macadamia nut is also a potent allergenic food. To date, there is little information about the allergenic proteins involved. In this study, using sera from macadamia nut allergic individuals, four IgE-binding proteins were detected. Their identities were determined by tandem mass spectrometry with de novo sequencing. Three IgE-reactive proteins, the vicilin Mac i 1, the legumin Mac i 2 and the antimicrobial peptide 2a/Mac i 1 (28–76) were purified from the nut while the non-specific lipid transfer protein was produced as a recombinant in Pichia pastoris. IgE-binding assays using sera from well-characterized groups of tree nut and/or peanut allergic patients revealed that the allergens were mainly recognized by sera from macadamia nut allergic individuals. Hence, these newly discovered allergens will enable molecular diagnostics to identify patients at high risk of macadamia nut allergy.

Study on novel PtNP-sorafenib and its interaction with VEGFR2.

With the developments of nanodrugs, some drugs have combined with nanoparticles (NPs) to reduce their side-effects and increase their therapeutic activities. Here, a novel nanodrug platinum nanoparticle-sorafenib (PtNP-SOR) was proposed for the first time. By means of molecular dynamics simulation, the stability and biocompatibility of PtNP-SOR were investigated. Then, the interaction mechanism between PtNP-SOR and vascular endothelial growth factor receptor 2 (VEGFR2) was explored and compared with that of the peptide 2a coated PtNPs. The results showed that PtNP-SOR could bind to VEGFR2 more stably, which was driven by the Coulombic and strong dispersion interaction between PtNP-SOR and VEGFR2. According to their contributions obtained from the decomposition of binding free energies, the key residues in VEGFR2 were identified to form the specific space, which increased the affinity with PtNP-SOR. This study provided useful insights to the design of PtNP-drugs as well as important theoretical proofs to the interaction between PtNP-SOR and VEGFR2 at a molecular level, which can be of large help during the development and optimization of novel nanodrugs.

Dietary nucleotides in the diets of on-growing grass carp (Ctenopharyngodon idella) suppress Aeromonas hydrophila induced intestinal inflammation and enhance intestinal disease-resistance via NF-κB and TOR signaling

The beneficial influences of nucleotides on fish intestinal immune responses and the mechanisms underlying the regulation of on-growing grass carp (Ctenopharyngodon idella) were evaluated using A. hydrophila induced intestinal inflammation model in vivo. Sixty days growth trial was carried out and fish were fed with mixed nucleotides diets: 0, 200.0, 400.0, 600.0, 800.0 and 1000.0 mg/kg diet. At the end of growth trial, grass carp were faced with infection trials with the challenge of A. hydrophila over a period of 14 days. Studies demonstrated that nucleotides supplementation had a powerful effect on inhibition of enteritis morbidity and inflammation caused by A. hydrophila in fish intestine. Dietary nucleotides elevated the production of intestinal acid phosphatase, lysozyme (LZ) activities, complements, immunoglobulin M and increased intestinal antimicrobial peptides (hepcidin, β-defensin-1, liver expressed antimicrobial peptide 2A and 2B) and mucin2 [only in distal intestine (DI)] gene expression. Nucleotides inhibited intestinal pro-inflammatory cytokines mRNA levels [interferon γ2 (IFN-γ2), tumor necrosis factor α (TNF-α), IL-6, IL-15, IL-17D, IL-12p35 and IL-12p40], which were correlated with nuclear factor kappa B (NF-κB) signaling [IκB kinase (IKK-β and IKK-γ)/inhibitor of (κBα) (IκBα)/NF-kB (p65 and c-Rel)]. Nucleotides increased intestinal anti-inflammatory cytokines [IL-11, transforming growth factor β1 (TGF-β1), IL-4/13A and IL-4/13B] gene expression partly associated with target of rapamycin (TOR) signaling [TOR/ribosomal protein S6 kinases 1 (S6K1) and eIF4E-binding proteins (4E-BP1)]. In summary, dietary nucleotides could enhance intestinal immune responses of fish via the inhibition of NF-κB and the promotion of TOR signaling. Furthermore, based on the parameters of enteritis morbidity and LZ activity in proximal intestine (PI), middle intestine (MI) and DI, optimal nucleotides supplementation levels for on-growing grass carp were estimated to be 580.35, 622.53, 613.21 and 617.98 mg/kg diet, respectively.

Dietary phytic acid weakened the antimicrobial activity and aggravated the inflammatory status of head kidney, spleen and skin in on-growing grass carp (Ctenopharyngodon idella)

The present study aimed to explore the effects of phytic acid (PA) on the antimicrobial activity and inflammatory response in three immune organs (head kidney, spleen and skin) of on-growing grass carp (Ctenopharyngodon idella). To achieve this goal, we first conducted a 60-day growth trial by feeding fish with graded levels of PA (0, 0.8, 1.6, 2.4, 3.2 and 4.0%). Then, the fish were challenged with Aeromonas hydrophila for 6 days. Compared with the control group, the following results were obtained regarding supplementation with certain levels of PA in the diet. (1) There was an increase in skin haemorrhage and lesion morbidity in fish. (2) There was a decrease in activities or contents of immune factors, including lysozyme (LZ), complement 3 (C3), C4 and immunoglobulin M (IgM), and there was downregulation of gene expression levels of hepcidin, liver-expressed antimicrobial peptide 2A (LEAP-2A), LEAP-2B, and β-defensin-1 in immune organs. (3) There was upregulation in the gene expression of the following pro-inflammatory cytokines: tumour necrosis factor α (TNF-α), interleukin 1β (IL-1β) (except in the spleen), interferon γ2 (IFN-γ2), IL-6 (except in the spleen), IL-8, IL-12p40, IL-15 and IL-17D. These changes were partly related to the nuclear factor kappa B (NF-κB) signalling pathway, but downregulation of mRNA levels of anti-inflammatory cytokines (transforming growth factor β1 (TGF-β1), TGF-β2, IL-413/A, IL-413/B, IL-10 (except in the skin) and IL-11) occurred in a manner partially related to the target of rapamycin (TOR) signalling pathway. Finally, based on the broken-line analysis of skin haemorrhage and lesion morbidity and IgM content in the head kidney, the maximum tolerance levels of PA for on-growing grass carp (120.56–452.00 g) were estimated to be 1.79 and 1.31% of the diet, respectively.

Optimization of a 2A self-cleaving peptide-based multigene expression system for efficient expression of upstream and downstream genes in silkworm.

The multigene expression system is highly attractive to co-express multiple genes or multi-subunit complex-based genes for their functional studies, and in gene therapy and visual tracking of expressed proteins. However, the current multiple gene co-expression strategies usually suffer from severe inefficiency and unbalanced expression of multiple genes. Here, we report on an improved 2A self-cleaving peptide (2A)-based multigene expression system (2A-MGES), by introducing an optimized Kozak region (Ck) and altering the gene arrangement, both of which contributed to the efficient expression of two fluorescent protein genes in silkworm. By co-expressing DsRed and EGFP genes in insect cells and silkworms, the potent Ck was first found to improve the translation efficiency of downstream genes, and the expression of the flanking genes of 2A were improved by altering the gene arrangement in 2A-MGES. Moreover, we showed that combining Ck and an optimized gene arrangement in 2A-MGES could synergistically improve the expression of genes in the cell. Further, these two flanking genes, regulated by modified 2A-MGES, were further co-expressed in the middle silk gland and secreted into the cocoon, and both achieved efficient expression in the transgenic silkworms and their cocoons. These results suggested that the modified Ck-2A-MGES will be a potent tool for multiple gene expression, for studies of their functions, and their applications in insect species.

Dietary phosphorus deficiency caused alteration of gill immune and physical barrier function in the grass carp (Ctenopharyngodon idella) after infection with Flavobacterium columnare

Phosphorus (P) is an important and essential mineral for fish. To investigate the effects of dietary P on gill immune and physical barrier function in grass carp (Ctenopharyngodon idella), fish were fed six graded levels of dietary available P (0.95–8.75 g/kg diet) for 60 days, and then sampled 3 days after a challenge test (exposure to water containing Flavobacterium columnare; 1.0 × 108 colony-forming units (cfu)/ml for 3 h). P deficiency resulted in a decline in gill antimicrobial compound production, and liver-expressed antimicrobial peptide 2A (LEAP-2A), LEAP-2B, hepcidin and β-defensin-1 transcript abundance. P deficiency also decreased target of rapamycin transcript and protein expression and increased nuclear factor kappa B p65 mRNA expression, which because of their association with anti-inflammatory and pro-inflammatory cytokine gene expression, respectively, suggesting that P deficiency increased post-infection gill inflammation and could impair immune barrier function. Furthermore, P deficiency: (1) Enhanced Fas ligand /caspase-8 death receptor and Bcl-2associated X protein/apoptotic protease activating factor-1/caspase-9 mRNA expression (which is indicative of increased apoptosis). (2) Inhibited Kelch-like-ECH-associated protein 1b/NF-E2-related factor 2 mRNA expression, decreased the expression of antioxidant enzyme transcript levels and activities (with the exception of CuZnSOD mRNA expression), and increased ROS, malondialdehyde and protein carbonyl levels. This data suggests that protection against ROS-related damage was compromised with P deficiency. (3) Up-regulated myosin light chain kinase transcript levels, a response that was associated with the down-regulation zonula occludens 1 (but not ZO-2), occludin and claudin (except claudin-12) mRNA levels. This latter result suggesting that tight junction functional integrity (and thus, gill physical barrier function) was impaired. These data, combined with the fact that P deficiency led to opercular deformation and gill rot syndrome, suggest that insufficient dietary levels of this mineral (i.e. below ~5.75 mg/kg P) could increase the susceptibility of fishes to water borne pathogens.

Inhibition of EGFR Activation by Bivalent Ligands Based on a Cyclic Peptide Mimicking the Dimerization Arm Structure of EGFR.

The epidermal growth factor receptor (EGFR) is a receptor in the ErbB family, and is overexpressed in some cancer cells. Recent research has shown that, since clustering of the EGFR increases the possibility of its dimerization and activation, the dimerization state of the EGFR on the cell surface is important for the recognition of the EGFR. In case a bivalent inhibitor has an optimized linker length, the clusters of the EGFR could be recognized with high affinity and kinase activation, which depends on EGF, could be suppressed. Peptide 1, which is derived from the dimerization arm of the EGFR, has been found previously to inhibit autophosphorylation of the EGFR. In this study, bivalent ligands based on peptide 1 with linkers of poly(L-proline) or poly-[(glycine)4(L-serine)] have been designed and synthesized. Bivalent ligands with polyproline linkers could maintain the distance between the ligand moieties. The inhibitory activity of these bivalent ligands against EGFR autophosphorylation was measured and was found to increase as the linker enlarges up to a 15-mer proline linker. The inhibitory activity of a bivalent ligand 7b is significantly higher compared to the corresponding monomeric peptide 2a. This suggests that bivalent EGFR ligands with optimal and rigid linkers could recognize the clusters of the EGFR with higher affinity and suppress kinase activation involving EGF.

Increased sesqui- and triterpene production by co-expression of HMG-CoA reductase and biotin carboxyl carrier protein in tobacco (Nicotiana benthamiana)

Terpenoids are the most diverse natural products with many industrial applications and are all synthesized from simple precursors, isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). In plants, IPP is synthesized by two distinct metabolic pathways – cytosolic mevalonate (MVA) pathway for C15 sesquiterpene and C30 triterpene, and plastidic methylerythritol phosphate (MEP) pathway for C10 monoterpene and C20 diterpene. A number of studies have altered the metabolic gene expressions in either the MVA or MEP pathway to increase terpene production; however, it remains unknown if the alteration of the acetyl-CoA pool in plastid fatty acid biosynthesis can influence terpenoid flux. Here, we focused on the fact that acetyl-CoA is the precursor for both fatty acid biosynthesis in plastid and terpene biosynthesis in cytosol, and the metabolic impact of increased plastidic acetyl-CoA level on the cytosolic terpene biosynthesis was investigated. In tobacco leaf infiltration studies, the acetyl-CoA carboxylase complex (the enzyme supplying malonyl-CoA in plastid) was partially inhibited by overexpressing the inactive form of biotin carboxyl carrier protein (BCCP) by a negative dominant effect. Overexpression of BCCP showed 1.4–2.4-fold increase of sesquiterpenes in cytosol; however, surprisingly overexpression of BCCP linked to truncated HMG-CoA reductase (tHMGR) by a cleavable peptide 2A showed 20–40-fold increases of C15 sesquiterpenes (α-bisabolol, amorphadiene, and valerenadiene) and a 6-fold increase of C30 β-amyrin. α-Bisabolol and β-amyrin production reached 28.8 mg g-1 and 9.8 mg g-1 dry weight, respectively. Detailed analyses showed that a large increase in flux was achieved by the additive effect of BCCP- and tHMGR-overexpression, and an enhanced tHMGR activity by 2A peptide tag. Kinetic analyses showed that tHMGR-2A has a three-fold higher kcat value than tHMGR. The tHMGR-2A-BCCP1 co-expression strategy in this work provides a new insight into metabolic cross-talks and can be a generally applicable approach to over-produce sesqui- and tri-terpene in plants.

Dietary myo-inositol deficiency decreased intestinal immune function related to NF-κB and TOR signaling in the intestine of young grass carp (Ctenopharyngodon idella)

In this study, we investigated the effects of dietary myo-inositol on the intestinal immune barrier function and related signaling pathway in young grass carp (Ctenopharyngodon idella). A total of 540 young grass carp (221.33 ± 0.84 g) were fed six diets containing graded levels of myo-inositol (27.0, 137.9, 286.8, 438.6, 587.7 and 737.3 mg/kg) for 10 weeks. After the growth trial, fish were challenged with Aeromonas hydrophila. The results indicated that compared with the optimal dietary myo-inositol level, myo-inositol deficiency (27.0 mg/kg diet): (1) decreased lysozyme (LZ) and acid phosphatase (ACP) activities, as well as complement 3 (C3), C4 and immunoglobulin M (IgM) contents in the proximal intestine (PI), middle intestine (MI) and distal intestine (DI) of young grass carp (P < 0.05). (2) down-regulated the mRNA levels of anti-microbial substance: liver expressed antimicrobial peptide (LEAP) 2A, LEAP-2B, hepcidin, β-defensin-1 and mucin2 in the PI, MI and DI of young grass carp (P < 0.05). (3) up-regulated pro-inflammatory cytokines [IL-1β (not in DI), TNF-α and IL-8], nuclear factor kappa B P65 (not NF-κB P52), c-Rel, IκB kinaseα (IKKα), IKKβ and IKKγ mRNA levels in the PI, MI and DI of young grass carp (P < 0.05); and down-regulated pro-inflammatory cytokines IL-15 (not in DI) and inhibitor of κBα (IκBα) mRNA levels (P < 0.05). (4) down-regulated the mRNA levels of anti-inflammatory cytokines [IL-10 (not in DI), IL-11, IL-4/13B (not IL-4/13A), TGF-β1 and TGF-β2], target of rapamycin (TOR), eIF4E-binding proteins 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6k1) in the PI, MI and DI of young grass carp (P < 0.05). All data indicated that myo-inositol deficiency could decrease fish intestine immunity and cause inflammation under infection of A. hydrophila. Finally, the optimal dietary myo-inositol levels for the ACP and LZ activities in the DI were estimated to be 415.1 and 296.9 mg/kg diet, respectively.

Administration of anabolic steroid during adolescence induces long-term cardiac hypertrophy and increases susceptibility to ischemia/reperfusion injury in adult Wistar rats

Chronic administration of anabolic androgenic steroids (AAS) in adult rats results in cardiac hypertrophy and increased susceptibility to myocardial ischemia/reperfusion (IR) injury. Molecular analyses demonstrated that hyperactivation of type 1 angiotensin II (AT1) receptor mediates cardiac hypertrophy induced by AAS and also induces down-regulation of myocardial ATP-sensitive potassium channel (KATP), resulting in loss of exercise-induced cardioprotection. Exposure to AAS during adolescence promoted long-term cardiovascular dysfunctions, such as dysautonomia. We tested the hypothesis that chronic AAS exposure in the pre/pubertal phase increases the susceptibility to myocardial ischemia/reperfusion (IR) injury in adult rats. Male Wistar rats (26day old) were treated with vehicle (Control, n=12) or testosterone propionate (TP) (AAS, 5mgkg−1 n=12) 5 times/week during 5 weeks. At the end of AAS exposure, rats underwent 23days of washout period and were submitted to euthanasia. Langendorff-perfused hearts were submitted to IR injury and evaluated for mechanical dysfunctions and infarct size. Molecular analysis was performed by mRNA levels of α-myosin heavy chain (MHC), βMHC and brain-derived natriuretic peptide (BNP), ryanodine receptor (RyR2) and sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) by quantitative RT-PCR (qRT-PCR). The expression of AT1 receptor and KATP channel subunits (Kir6.1 and SURa) was analyzed by qRT-PCR and Western Blot. NADPH oxidase (Nox)-related reactive oxygen species generation was assessed by spectrofluorimetry. The expression of antioxidant enzymes was measured by qRT-PCR in order to address a potential role of redox unbalance. AAS exposure promoted long-term cardiac hypertrophy characterized by increased expression of βMHC and βMHC/αMHC ratio. Baseline derivative of pressure (dP/dt) was impaired by AAS exposure. Postischemic recovery of mechanical properties was impaired (decreased left ventricle [LV] developed pressure and maximal dP/dt; increased LV end-diastolic pressure and minimal dP/dt) and infarct size was larger in the AAS group. Catalase mRNA expression was significantly decreased in the AAS group. In conclusion, chronic administration of AAS during adolescence promoted long-term pathological cardiac hypertrophy and persistent increase in the susceptibility to myocardial IR injury possible due to disturbances on catalase expression.

Effect of B‐type natriuretic peptide and phosphodiesterase 2A is coupled to neurotransmitter release in pro‐hypertensive rats

BackgroundB‐type natriuretic peptide (BNP) decreases cardiac sympathetic neurotransmission by attenuating activation of neuronal calcium channels and the intracellular calcium transient via a cGMP‐protein kinase G (PKG) pathway. Elevated levels of BNP are regarded as an early compensatory response to cardiac myocyte hypertrophy, although exogenously administered BNP shows poor clinical efficacy in heart failure and hypertension. Emerging evidence suggests that phosphodiesterase 2A (PDE2A) is upregulated in heart failure. Therefore we tested whether PDE2A was directly involved in modulating cardiac neurotransmitter release and Ca2+ handling from young (pre‐hypertensive) spontaneously hypertensive rats (SHRs) that show an enhanced Ca2+ phenotype.Methods and ResultsFour week old SHRs and the normotensive Wistar‐Kyoto (WKY) rats were humanely killed by an approved Home Office (UK) schedule 1 method. Intracellular free Ca2+ concentration ([Ca2+]i) was measured by ratiometric fluorescence imaging using fura‐2AM in cultured cardiac sympathetic neurons. The evoked [Ca2+]i transient was evaluated following 30 sec exposure to 50 mM KCL in the Tyrode solution. [3H] labelled noradrenaline (NE) release was measured from isolated spontaneously beating atrial in response to 5Hz field stimulation for 1 minute at the 16th (S1, control) and 40th (S2, with BNP) minutes. BNP significantly reduced the magnitude of the calcium transients in WKY rats, but not in SHR. PDE2A protein and activity levels from the stellate ganglia were greater in SHRs (n=8) compared with WKY rats (n=8). PDE2 inhibitor Bay60‐7550 restored the capacity of BNP to reduce [Ca2+]i in the SHR. Overexpression of dnPDE2A (a catalytically‐dead mutant of PDE2A) using a viral vector (Ad.CMVmCherry. dnPDE2A) also rescued the BNP inhibition of the calcium transients from the SHR. Functionally, BNP significantly reduced [3H]‐NE release in the WKY, whereas it failed in the SHR. Overexpression of dnPDE2A recovered the BNP inhibition of the NE release in the SHR.ConclusionThese data support the hypothesis that higher PDE2A expression in the SHR abolishes the capacity of BNP to reduce neurotransmitters release. They also suggest that inhibiting neuronal PDE2 may be important in restoring the efficacy of BNP to decrease sympathetic neurotransmission in cases of dysautonomia.Support or Funding InformationSupported by the British Heart Foundation

2A self-cleaving peptide-based multi-gene expression system in the silkworm Bombyx mori.

Fundamental and applied studies of silkworms have entered the functional genomics era. Here, we report a multi-gene expression system (MGES) based on 2A self-cleaving peptide (2A), which regulates the simultaneous expression and cleavage of multiple gene targets in the silk gland of transgenic silkworms. First, a glycine-serine-glycine spacer (GSG) was found to significantly improve the cleavage efficiency of 2A. Then, the cleavage efficiency of six types of 2As with GSG was analyzed. The shortest porcine teschovirus-1 2A (P2A-GSG) exhibited the highest cleavage efficiency in all insect cell lines that we tested. Next, P2A-GSG successfully cleaved the artificial human serum albumin (66 kDa) linked with human acidic fibroblast growth factor (20.2 kDa) fusion genes and vitellogenin receptor fragment (196 kD) of silkworm linked with EGFP fusion genes, importantly, vitellogenin receptor protein was secreted to the outside of cells. Furthermore, P2A-GSG successfully mediated the simultaneous expression and cleavage of a DsRed and EGFP fusion gene in silk glands and caused secretion into the cocoon of transgenic silkworms using our sericin1 expression system. We predicted that the MGES would be an efficient tool for gene function research and innovative research on various functional silk materials in medicine, cosmetics, and other biomedical areas.

Effect of B-type natriuretic peptide and phosphodiesterase 2A is coupled to neurotransmitter release in pro-hypertensive rats

Effect of B-type natriuretic peptide and phosphodiesterase 2A is coupled to neurotransmitter release in pro-hypertensive rats

2A and the Auxin-Based Degron System Facilitate Control of Protein Levels in Plasmodium falciparum

Analysis of gene function in Plasmodium falciparum, the most important human malaria parasite, is restricted by the lack of robust and simple reverse genetic tools. Approaches to manipulate protein levels post-translationally are powerful tools to study protein-off effects especially in the haploid malaria parasite where genetic knockouts of essential genes are lethal. We investigated if the auxin-inducible degron system is functional in P. falciparum and found that degron-tagged yellow fluorescent protein levels were efficiently reduced upon addition of auxin which otherwise had no effect on parasite viability. The genetic components required in this conditional approach were co-expressed in P. falciparum by applying the small peptide 2A. 2A is a self-processing peptide from Foot-And-Mouth Disease virus that allows the whole conditional system to be accommodated on a single plasmid vector and ensures stoichiometric expression levels.

Processing of the VP1/2A Junction Is Not Necessary for Production of Foot-and-Mouth Disease Virus Empty Capsids and Infectious Viruses: Characterization of “Self-Tagged” Particles

The foot-and-mouth disease virus (FMDV) capsid protein precursor, P1-2A, is cleaved by 3C(pro) to generate VP0, VP3, VP1, and the peptide 2A. The capsid proteins self-assemble into empty capsid particles or viruses which do not contain 2A. In a cell culture-adapted strain of FMDV (O1 Manisa [Lindholm]), three different amino acid substitutions (E83K, S134C, and K210E) were identified within the VP1 region of the P1-2A precursor compared to the field strain (wild type [wt]). Expression of the O1 Manisa P1-2A (wt or with the S134C substitution in VP1) plus 3C(pro), using a transient expression system, resulted in efficient capsid protein production and self-assembly of empty capsid particles. Removal of the 2A peptide from the capsid protein precursor had no effect on capsid protein processing or particle assembly. However, modification of E83K alone abrogated particle assembly with no apparent effect on protein processing. Interestingly, the K210E substitution, close to the VP1/2A junction, completely blocked processing by 3C(pro) at this cleavage site, but efficient assembly of "self-tagged" empty capsid particles, containing the uncleaved VP1-2A, was observed. These self-tagged particles behaved like the unmodified empty capsids in antigen enzyme-linked immunosorbent assays and integrin receptor binding assays. Furthermore, mutant viruses with uncleaved VP1-2A could be rescued in cells from full-length FMDV RNA transcripts encoding the K210E substitution in VP1. Thus, cleavage of the VP1/2A junction is not essential for virus viability. The production of such engineered self-tagged empty capsid particles may facilitate their purification for use as diagnostic reagents and vaccines.

Differentiation at the MHCIIα and Cath2 loci in sympatric Salvelinus alpinus resource morphs in Lake Thingvallavatn.

Northern freshwater fish may be suitable for the genetic dissection of ecological traits because they invaded new habitats after the last ice age (∼10.000 years ago). Arctic charr (Salvelinus alpinus) colonizing streams and lakes in Iceland gave rise to multiple populations of small benthic morphotypes, often in sympatry with a pelagic morphotype. Earlier studies have revealed significant, but subtle, genetic differentiation between the three most common morphs in Lake Thingvallavatn. We conducted a population genetic screen on four immunological candidate genes Cathelicidin 2 (Cath2), Hepcidin (Hamp), Liver expressed antimicrobial peptide 2a (Leap-2a), and Major Histocompatibility Complex IIα (MHCIIα) and a mitochondrial marker (D-loop) among the three most common Lake Thingvallavatn charr morphs. Significant differences in allele frequencies were found between morphs at the Cath2 and MHCIIα loci. No such signal was detected in the D-loop nor in the other two immunological genes. In Cath2 the small benthic morph deviated from the other two (FST = 0.13), one of the substitutions detected constituting an amino acid replacement polymorphism in the antimicrobial peptide. A more striking difference was found in the MHCIIα. Two haplotypes were very common in the lake, and their frequency differed greatly between the morphotypes (from 22% to 93.5%, FST = 0.67). We then expanded our study by surveying the variation in Cath2 and MHCIIα in 9 Arctic charr populations from around Iceland. The populations varied greatly in terms of allele frequencies at Cath2, but the variation did not correlate with morphotype. At the MHCIIα locus, the variation was nearly identical to the variation in the two benthic morphs of Lake Thingvallavatn. The results are consistent with a scenario where parts of the immune systems have diverged substantially among Arctic charr populations in Iceland, after colonizing the island ∼10.000 years ago.