Journal: BMC Genomics

Article Title: Evolutionarily recent, insertional fission of mitochondrial cox2 into complementary genes in bilaterian Metazoa

doi: 10.1186/s12864-017-3626-5

Figure Lengend Snippet: Putative polypeptides encoded by the cox2 -splitting DNA insert in the C. p. fossulana mitogenome. a Western blot and ribbon diagram of the I-TASSER-modeled three-dimensional structure of the QNU (the larger of its two isoforms) polypeptide. The tertiary structure was predicted by combining de novo and locally applied template-based modeling (PDB templates for local structure predictions were: 1wOrA, 3iymA, 2ocwA, 1pclA, 3cm9S). Signature motif and regions with similarity to nucleic acid-interacting proteins (Nai) and the active site of HNH homing endonucleases (HNH) are indicated on the polypeptide linear model. The inset shows the nuclease activity assay of the recombinant QNU using plasmid DNA as substrate, analyzed by agarose gel electrophoresis. No plasmid degradation was observed in the absence of recombinant proteins (P mock). The addition of rQNU caused a decrease in both SC and C forms of the plasmid and smearing of the L form, indicating at least endonuclease activity of the recombinant QNU (+rQNU). Addition of rΔQNU had no effect on the level of any form of the plasmid, indicating the absence of nuclease activity (+rΔQNU) over a 2-h incubation at 37 °C. Plasmid topology: SC, supercoil; L, linear; C, coil. Deletion of Gln-Asn (QN) repeats suppressed the nuclease activity of the rQNU polypeptide. b Western blot of the putative WFW polypeptide and deduced sequence of the repetitive signature motif of WFW that was predicted to adopt helical structure stabilized by Trp residues

Article Snippet: Polyclonal Abs against synthetic epitopes of the C. p. fossulana polypeptides COXIIA, COXIIB, QNU, and WFW were raised in rabbits and affinity-purified (GenScript, Piscataway, NJ).

Techniques: Western Blot, Activity Assay, Recombinant, Plasmid Preparation, Agarose Gel Electrophoresis, Incubation, Sequencing

Journal: The Journal of Neuroscience

Article Title: Peripheral Antinociceptive Effects of Exogenous and Immune Cell-Derived Endomorphins in Prolonged Inflammatory Pain

doi: 10.1523/JNEUROSCI.4349-05.2006

Figure Lengend Snippet: Opioid receptor selectivity of exogenous EM-1- and EM-2-induced antinociception. Left, EM-1-induced antinociception was dose dependently blocked by μ-receptor (CTOP) but not δ-receptor (ICI 174,864) or κ-receptor (norBNI) selective antagonists (p < 0.001, ANOVA, linear regression; p > 0.05, ANOVA, respectively). Middle, EM-2-induced antinociception was dose dependently blocked by μ-receptor (CTOP) and attenuated by δ-receptor (ICI 174,864 and naltrindole) selective antagonists (p < 0.001, ANOVA, linear regression). EM-2-induced antinociception was not significantly changed by a κ-receptor (norBNI) selective antagonist (p > 0.05, ANOVA). Right, EM-2-induced antinociception was not significantly changed by antibodies against Met-enkephalin (anti-Met-ENK), Leu-enkephalin (anti-Leu-ENK), and β-endorphin (anti-END) (p > 0.05, ANOVA). Receptor antagonists and peptide antibodies were coadministered intraplantarly into inflamed paws with either EM-1 or EM-2. PPTs were measured 5 min after injections. Dashed lines represent baseline paw-pressure thresholds of representative groups and are 40 ± 1.2 g. Data are expressed as means ± SEM.

Article Snippet: The sections were then incubated overnight with rabbit anti-rat polyclonal Abs against Leu-enkephalin (4 μg/ml; Phoenix Pharmaceuticals), β-endorphin (1:1000; Peninsula Laboratories), EM-1, or EM-2 (7 μg/ml; Chemicon, Hampshire, UK).

Techniques:

Journal: ImmunoHorizons

Article Title: Alboserpin, the Main Salivary Anticoagulant from the Disease Vector Aedes albopictus , Displays Anti–FXa-PAR Signaling In Vitro and In Vivo

doi: 10.4049/immunohorizons.2200045

Figure Lengend Snippet: HDMVECns were left untreated (control), treated with Alboserpin (50 nM) or FXa (50 nM), or Alboserpin:FXa for 3 h. (A) Cell supernatants were used for cytokine and chemokine Luminex ELISA. (B) Gene expression of PARs in HDMVECns. Data are shown as relative quantification versus control (resting cells). (C) ERK1/2 protein expression was analyzed by Western blot. Cell lysates were analyzed for levels of p-ERK, and ERK (total ERK) and GAPDH was used as a loading control. ( D ) The ratio of p-ERK/ERK is presented in arbitrary units (A.U). ( E ) ERK1/2 protein expression was analyzed using In-Cell Western blots (ICW). ( F ) Intensity ratio (p-ERK/ERK). ( G and H ) NF-κB gene expression and protein expression were analyzed using RT-PCR and Western blots, respectively. (I and J) ICAM and VCAM gene expression were analyzed using RT-PCR. Data are shown as relative quantification versus control (resting cells). Data from three independent experiments performed in triplicate are plotted. Error bars indicate SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Polyclonal Abs against Alboserpin were raised in rabbits by Noble Life Sciences (Woodbine) using a standard protocol.

Techniques: Control, Luminex, Enzyme-linked Immunosorbent Assay, Gene Expression, Quantitative Proteomics, Expressing, Western Blot, In-Cell ELISA, Reverse Transcription Polymerase Chain Reaction

Journal: ImmunoHorizons

Article Title: Alboserpin, the Main Salivary Anticoagulant from the Disease Vector Aedes albopictus , Displays Anti–FXa-PAR Signaling In Vitro and In Vivo

doi: 10.4049/immunohorizons.2200045

Figure Lengend Snippet: Representative data of FXa cleavage on PAR-2 peptide was investigated based on fluorescence resonance energy transfer (FRET) technology. Peptide sequence that spans the cleavage sites for mouse PAR-2 (NSKGRSLIGR) was synthesized with the fluorescent group Mca (7-methoxycoumarin-4-acetic acid) and the quenching group Dnp (2,4-DNP) at the N- and C-terminal ends, respectively. FXa (10 nM) was incubated with different concentrations of purified Alboserpin (0–200 nM) at 37°C in 20mMTris–HCl, 150 mM NaCl, Tween 20 0.01% (pH 7.4). After 15-min incubation, peptide was added in a 100 μl final reaction volume. The peptide hydrolysis rate was followed at 320 nm excitation and 420 nm emission in kinetic mode at 30°C in a microplate reader

Article Snippet: Polyclonal Abs against Alboserpin were raised in rabbits by Noble Life Sciences (Woodbine) using a standard protocol.

Techniques: Fluorescence, Förster Resonance Energy Transfer, Sequencing, Synthesized, Incubation, Purification

Journal: ImmunoHorizons

Article Title: Alboserpin, the Main Salivary Anticoagulant from the Disease Vector Aedes albopictus , Displays Anti–FXa-PAR Signaling In Vitro and In Vivo

doi: 10.4049/immunohorizons.2200045

Figure Lengend Snippet: Effect of Alboserpin in paw edema assay triggered by FXa. ( A ) Footpads of C3H/HeJ mice were intradermally injected with 30 μl of FXa (50 nM) alone or incubated along with 50 nM Alboserpin. As a control, 30 μl of PBS or 30 ml of 1 mg Alboserpin was injected. Formation of edema (increasein paw thickness in millimeters) was measured using a caliper before injection of FXa or after 15, 30, 45, and 60 min of injections. Data represent SEM of five footpads per experimental group. ( B ) Effect of Alboserpin in FXa-induced inflammatory cytokine and chemokine. Cytokine and chemokine levels in paw tissue extracts after 60 min of intradermal injection were determined by Luminex ELISA assay. Data from three independent experiments performed in triplicate are plotted. Error bars indicate SEM of eight footpads per experimental group. Error bars indicate SEM. ** p < 0.01, **** p < 0.0001. ns, not significant.

Article Snippet: Polyclonal Abs against Alboserpin were raised in rabbits by Noble Life Sciences (Woodbine) using a standard protocol.

Techniques: Injection, Incubation, Control, Luminex, Enzyme-linked Immunosorbent Assay

Journal: ImmunoHorizons

Article Title: Alboserpin, the Main Salivary Anticoagulant from the Disease Vector Aedes albopictus , Displays Anti–FXa-PAR Signaling In Vitro and In Vivo

doi: 10.4049/immunohorizons.2200045

Figure Lengend Snippet: Endothelial cell monolayer permeability assay using FITC-dextran. ( A ) Crystal violet staining of the HDMVECn monolayer pretreated with Alboserpin and FXa at 50 nM for 4 h. ( B ) The fluorescence intensity of FITC-conjugated dextran leaking from the upper to the lower chambers was measured at different time points after treatment of HDMVECns with either FXa alone, Alboserpin, or Alboserpin along with FXa. Data from three independent experiments performed in duplicate are plotted. Error bars indicate SEM. ( C and D ) Schematic representation of the sequential steps of the Miles assay in the mouse and corresponding inoculations in different locations in skin biopsies. ( E ) Spectrophotometric analysis (610 nm) of vascular leaked formamide-extracted Evans blue dye content in the skin injected with 50 μM Alboserpin, FXa, and both FXa-Alboserpin at the same concentration. PBS was used as a negative control. * p < 0.05, *** p < 0.001, **** p < 0.0001. ns, not significant.

Article Snippet: Polyclonal Abs against Alboserpin were raised in rabbits by Noble Life Sciences (Woodbine) using a standard protocol.

Techniques: Permeability, Staining, Fluorescence, Injection, Concentration Assay, Negative Control

Journal: ImmunoHorizons

Article Title: Alboserpin, the Main Salivary Anticoagulant from the Disease Vector Aedes albopictus , Displays Anti–FXa-PAR Signaling In Vitro and In Vivo

doi: 10.4049/immunohorizons.2200045

Figure Lengend Snippet: During a mosquito bite, tissue injury causes the initiation of signaling through the extrinsic and intrinsic pathways that leads to the formation of FXa. FXa further converts prothrombin to thrombin, leading to clot formation. FXa triggers an inflammatory pathway in the endothelial cells by activating PAR receptors, ICAM and VCAM adhesion molecule expression, ERK1/2 signaling, NF-κB signaling, secretion of inflammatory cytokines, and disruption of endothelial cell barrier permeability. Mosquito A. albopictus salivary gland protein Alboserpin, a highly specific, strong inhibitor of FXa, prevents cleavage of PAR-2 by FXa, resulting in strong anti-inflammatory activity in vitro and in vivo.

Article Snippet: Polyclonal Abs against Alboserpin were raised in rabbits by Noble Life Sciences (Woodbine) using a standard protocol.

Techniques: Expressing, Disruption, Permeability, Activity Assay, In Vitro, In Vivo