Surface plasmon resonance imaging protein arrays for analysis of triple protein interactions of HPV, E6, E6AP, and p53

We have developed a surface plasmon resonance (SPR)-based protein microarray to study protein-protein interactions in a high-throughput mode. As a model system, triple protein interactions have been explored with human papillomaviral E6 protein, tumor suppressor p53, and ubiquitin ligase E6AP. Human papillomavirus (HPV) is known to be a causative agent of cervical cancer. Upon infection, the viral E6 protein forms a heterotrimeric protein complex with p53 and E6AP. The formation of the complex eventually results in the degradation of p53. In the present study, a GST-fused E6AP protein was layered onto a glutathione (GSH)-modified gold chip surface. The specific binding of GST-E6AP protein onto the gold chip surface was facilitated through the affinity of GST to its specific ligand GSH. The interacting proteins (E6 and/or p53) were then spotted. Detection of the interaction was performed using a SPR imaging (SPRI) technique. The resulting SPRI intensity data showed that the protein-protein interactions of E6AP, E6, and p53 were detected in a concentration-dependent manner, suggesting that the SPRI-based microarray system can be an effective tool to study protein-protein interactions where multiple proteins are involved.     

Protein expression changes in human monocytic THP-1 cells treated with lipoteichoic acid from Lactobacillus plantarum and Staphylococcus aureus

Lipoteichoic acid (LTA) from Staphylococcus aureus (aLTA) and from Lactobacillus plantarum LTA (pLTA) are both recognized by Toll-like receptor 2 (TLR2), but cause different stimulatory effects on the innate immune and inflammatory responses, and their underlying cellular mechanisms are unknown. In this study, comparative proteome analysis was performed using two-dimensional gel electrophoresis and mass spectrometry on protein extracts from human monocyte THP-1 cells stimulated with either aLTA or pLTA. Differentially expressed proteins might be involved in innate immunity and inflammation. Cells treated with aLTA and with pLTA showed different protein expression profiles. Of 60 identified proteins, 10 were present only in treated cells (8 in aLTA-treated only, and 2 in pLTA-treated only), 1 protein (IMPDH2) was suppressed by pLTA, and 49 were up- or down-regulated more than three-fold by aLTA- or pLTA- stimulation. Several proteins involved in immunity or inflammation, antioxidation, or RNA processing were significantly changed in expression by aLTA- or pLTA-stimulation, including cyclophilin A, HLA-B27, D-dopachrome tautomerase, Mn- SOD, hnRNP-C, PSF and KSRP. These data demonstrated that aLTA and pLTA had different effects on the protein profile of THP-1 cells. Comparison of the proteome alterations will provide candidate biomarkers for further investigation of the immunomodulatory effects of aLTA and pLTA, and the involvement of aLTA in the pathogenesis of Staphylococcus aureus sepsis.     

Dishevelled mediates ephrinB1 signalling in the eye field through the planar cell polarity pathway

An important step in retinal development is the positioning of progenitors within the eye field where they receive the local environmental signals that will direct their ultimate fate. Recent evidence indicates that ephrinB1 functions in retinal progenitor movement, but the signalling pathway is unclear. We present evidence that ephrinB1 signals through its intracellular domain to control retinal progenitor movement into the eye field by interacting with Xenopus Dishevelled (Xdsh), and by using the planar cell polarity (PCP) pathway. Blocking Xdsh translation prevents retinal progeny from entering the eye field, similarly to the morpholino-mediated loss of ephrinB1 (ref. 2). Overexpression of Xdsh can rescue the phenotype induced by loss of ephrinB1, and this rescue (as well as a physical association between Xdsh and ephrinB1) is completely dependent on the DEP (Dishevelled, Egl-10, Pleckstrin) domain of Xdsh. Similar gain- and loss-of-function experiments suggest that Xdsh associates with ephrinB1 and mediates ephrinB1 signalling through downstream members of the PCP pathway during eye field formation.     

Lipase inhibitory activity of ethyl acetate fraction from Ecklonia cava extracts

Obesity is a key contributing risk factor to cardiovascular disease, certain cancers, and diabetes. Much effort has being made to investigate potential inhibitors against lipase from natural products. The ethyl acetate (EA) extract of Ecklonia cava (EC) were tested for their ability to inhibit pancreatic lipase activity in vitro. The 22 sub-fractions from EA extract were separated using silica gel column chromatography. Among the sub-fractions, the EA6 sub-fraction exhibited the highest inhibitory activity. Dieckol compound was isolated from the EA6 sub-fraction, which inhibited the lipase activity in a concentrationdependent manner with IC₅₀ value at 0.26 mg/mL. These results suggest that EC has potential as a natural antiobesity agent.     

High-level expression of an antimicrobial peptide histonin as a natural form by multimerization and furin-mediated cleavage

Direct expression of an antimicrobial peptide (AMP) in Escherichia coli causes several problems such as the toxicity of AMP to the host cell, its susceptibility to proteolytic degradation, and decreased antimicrobial activity due to the additional residue(s) introduced after cleavage of AMPs from fusion partners. To overcome these problems and produce a large quantity of a potent AMP histonin (RAGLQFPVGKLLKKLLKRLKR) in E. coli, an efficient expression system was developed, in which the toxicity of histonin was neutralized by a fusion partner F4 (a truncated fragment of PurF protein) and the productivity was increased by a multimeric expression of a histonin gene. The expression level of the fusion proteins reached a maximum with a 12-mer of a histonin gene. In addition, because of the RLKR residues present at the C terminus of histonin, furin cleavage of the multimeric histonin expressed produces an intact, natural histonin. The AMP activity of the histonin produced in E. coli was identical to that of a synthetic histonin. With our expression system, 167 mg of histonin was obtained from 1 l of E. coli culture. These results may lead to a cost-effective solution for the mass production of AMPs that are toxic to a host.     

Genus delimitation,biogeography and diversification of Choristoneura Lederer (Lepidoptera: Tortricidae) based on molecular evidence

Widely known for pest species that include major modulators of temperate forests, the genus Choristoneura is part of the species‐rich tribe Archipini of leafroller moths (Tortricidae). Delimitation of the genus has remained unresolved because no phylogeny has included species endemic to Africa and studies have often omitted the type species of the genus. Further taxonomic confusion has been generated by the transfer of Archips occidentalis (Walsingham) to Choristoneura, creating a homonym with Choristoneura occidentalis Freeman, an important defoliator of North American forests. To define the limits of the genus, we reconstructed a phylogeny using DNA sequences for mitochondrial cytochrome oxidase subunit I and nuclear ribosomal 28S genes. Our ingroup included 23 Choristoneura species‐level taxa, complemented by a large sample of outgroups comprising 82 species of Archipini and other Tortricidae. We generated a time‐calibrated tree using fossil and secondary calibrations and we inferred biogeographic and diversification processes in Choristoneura. Our analysis recovered the genus as polyphyletic, with Archips occidentalis, Choristoneura simonyi and Choristoneura evanidana excluded from the main clade. Based on the recovered phylogenies and a redefinition, we restrict Choristoneura primarily to species with a northern hemisphere distribution. Our analysis supports A. occidentalis as the sister group of Cacoecimorpha pronubana, C. simonyi as the sister of ‘Xenotemna’ pallorana, and C. evanidana as the sister of Archips purpurana. A new combination is proposed: Archips evanidana comb.n. ; the availability of ‘Xenotemna’ as a valid name is discussed and A. occidentalis is considered as an orphaned name within the Archipini. We found support for a Holarctic origin of Choristoneura about 23 Ma, followed by early divergence in the Palearctic region. The main divergence occurred at 16 Ma, with one clade in the Nearctic and another in the Palearctic. Subsequent cladogenetic events were synchronous and related to herbivorous specialization, with each clade divided into coniferophagous and polyphagous lineages. Their specialization as conifer feeders temporally matched the expansion of boreal forest during the Miocene.     

Novel tacrine-pyridinium hybrid reactivators of organophosphorus-inhibited acetylcholinesterase: Synthesis,molecular docking,and in vitro reactivation study

First-line medical treatment against nerve agents consists of co-administration of anticholinergic agents and oxime reactivators, which reactivate inhibited AChE. Pralidoxime, a commonly used oxime reactivator, is effective against some nerve agents but not against others; thus, new oxime reactivators are needed. Novel tacrine-pyridinium hybrid reactivators in which 4-pyridinealdoxime derivatives are connected to tacrine moieties by linear carbon chains of different lengths (C2–C7) were prepared (Scheme 1, 5a–f). Their binding affinities to electric eel AChE were tested because oximes can inhibit free AChE, and the highest AChE activity (95%, 92%, and 90%) was observed at 1?μM concentrations of the oximes (5a, 5b, and 5c, respectively). Based on their inhibitory affinities towards free AChE, 1?μM concentrations of the oxime derivatives (5) were used to examine reactivation of paraoxon-inhibited AChE. Reactivation ability increased as the carbon linker chains lengthened (n?=?2–5), and 5c and 5d showed remarkable reactivation ability (41%) compared to that of 2-PAM (16%) and HI-6 (4%) against paraoxon-inhibited electric eel AChE at 1?μM concentrations. Molecular docking simulation showed that the most stable binding free energy was observed in 5c at 73.79?kcal?mol^?1, and the binding mode of 5c is acceptable for the oxygen atom of oximate to attack the phosphorus atom of paraoxon and reactivate paraoxon-inhibited eel AChE model structure.     

Surface Plasmon Resonance Imaging-Based Protein Array Chip System for Monitoring a Hexahistidine-Tagged Protein during Expression and Purification

A surface plasmon resonance imaging-based Ni²⁺-iminodiacetic acid-coated gold chip system was developed to enable specific detection of a hexahistidine-tagged recombinant protein in crude extracts or in column chromatography fractions. This system is especially advantageous for high-throughput analysis of multiple proteins.     

Effects of Panax ginseng on the nerve growth factor expression in testosterone induced benign prostatic hyperplasia

The prostatic hyperplasia in benign prostatic hyperplasia (BPH) leads to obstructive micturition symptoms. Previous studies showed that pontine micturition center (PMC), ventrolateral periaqueductal gray (vlPAG), and medial preopticnucleus (MPA) regions in the brain have been known to regulate the urinary bladder function. The present study shows the influences of Panax ginseng on nerve growth factor (NGF) expressions in PMC, vlPAG, and MPA regions in the brain. Wistar rats were used for the present study. The rats split into four groups; 4 groups (n = 6) in control group, BPH-induced group, BPH-induced and P. ginseng-treated group, and BPH-induced and finasteride-treated group. BPH in rats was induced by testosterone and the animals were evaluated for NGF expression in PMC, vlPAG, and MPA regions in the brain. The NGF expression was identified using immunohistochemistry (IHC). The NGF expression by IHC showed spots with dark brown color. In our results, NGF expressions in PMC, vlPAG, and MPA regions in the brainstem of the BPH-induced group showed increase than the control animal. These increased NGF expressions in three regions were decreased using treatment with P. ginseng (200 mg/kg). These results suggest that P. ginseng has therapeutic effects on the symptoms of BPH and is associated with the regulation of NGF expression in the brain. In conclusion, the administration of P. ginseng helps nerve growth factor activation.