Simultaneous voltammetric determination of norepinephrine, ascorbic acid and uric acid on polycalconcarboxylic acid modified glassy carbon electrode

A novel polycalconcarboxylic acid (CCA) modified glassy carbon electrode (GCE) was fabricated by electropolymerization and then successfully used to simultaneously determine ascorbic acid (AA), norepinephrine (NE) and uric acid (UA). The characterization of electrochemically synthesized Poly-CCA film was investigated by atomic force microscopy (AFM), electrochemical impedance spectroscopy (EIS) and voltammetric methods. It was found that the electrochemical behavior of the polymer-modified electrode depended on film thickness, i.e., the electropylmyerization time. Based on the electrochemical data, the charge transfer coefficient (alpha) and the surface coverage (Gamma) were calculated. This poly-CCA modified GCE could reduce the overpotential of ascorbic acid (AA), norepinephrine (NE) and uric acid (UA) oxidation in phosphate buffer solution (pH 6.0), while it increases the peak current significantly. The current peak separations of AA/NE, NE/UA and AA/UA on this modified electrode are 91mV, 256mV and 390mV in CV at 100mVs(-1), respectively. Therefore, the voltammetric responses of these three compounds can be well resolved on the polymer-modified electrode, and simultaneously determination of these three compounds can be achieved. In addition, this modified electrode can be successfully applied to determine AA and NE in injection and UA in urine samples without interferences.     

Analysis of the Intestinal Microflora in Hepialus gonggaensis Larvae Using 16S rRNA Sequences

Gut microbial diversity provides insight into the basic function of a gut microbial ecosystem. In this study, restriction fragment length polymorphism 16S rRNA sequences was used to detect the intestinal microbial diversity of Hepialus gonggaensis larvae. The total DNA of microorganisms was extracted from the intestinal contents and 16S rRNA was amplified. A nearly full-length of 16S rRNA sequence library was constructed. The fingerprints of the microorganisms were analyzed by isolating plasmid and then digesting them with EcoRI, MspI, and HaeIII enzymes, respectively. The library established includes 35 restriction endonuclease types and a phylogenetic tree depicted the linkage of the isolated microbial from the guts of H. gonggaensis larvae. The dominant bacteria in the guts of H. gonggaensis larvae belong to Rahnella sp and Carnobacterium sp and accounted for 45.58% and 30.88% of the total 16S rRNA clones library, respectively. The result showed that bacteria diversity in the guts of H. gonggaensis larvae had some differences from those isolated from normal environment.     

The role of oxidative stress in ambient particulate matter-induced lung diseases and its implications in the toxicity of engineered nanoparticles

Ambient particulate matter (PM) is an environmental factor that has been associated with increased respiratory morbidity and mortality. The major effect of ambient PM on the pulmonary system is the exacerbation of inflammation, especially in susceptible people. One of the mechanisms by which ambient PM exerts its proinflammatory effects is the generation of oxidative stress by its chemical compounds and metals. Cellular responses to PM-induced oxidative stress include activation of antioxidant defense, inflammation, and toxicity. The proinflammatory effect of PM in the lung is characterized by increased cytokine/chemokine production and adhesion molecule expression. Moreover, there is evidence that ambient PM can act as an adjuvant for allergic sensitization, which raises the possibility that long-term PM exposure may lead to increased prevalence of asthma. In addition to ambient PM, rapid expansion of nanotechnology has introduced the potential that engineered nanoparticles (NP) may also become airborne and may contribute to pulmonary diseases by novel mechanisms that could include oxidant injury. Currently, little is known about the potential adverse health effects of these particles. In this communication, the mechanisms by which particulate pollutants, including ambient PM and engineered NP, exert their adverse effects through the generation of oxidative stress and the impacts of oxidant injury in the respiratory tract will be reviewed. The importance of cellular antioxidant and detoxification pathways in protecting against particle-induced lung damage will also be discussed.     

Expression change of β-1,4 galactosyltransferase I, V mRNAs and Galβ1,4GlcNAc group in rat sciatic nerve after crush

Glycosylation is one of the most important post-translational modifications. It is clear that the single step of β-1,4-galactosylation is performed by a family of β-1,4-galactosyltransferases (β-1,4-GalTs), and that each member of this family may play a distinct role in different tissues and cells. β-1,4-GalT I and V are involved in the biosynthesis of N-linked oligosaccharides. In the present study, Real-time PCR revealed that the β-1,4-GalT I and V mRNAs reached peaks at 2 w after sciatic nerve crush. In situ hybridization showed that at 1 d after sciatic nerve crush, the expression levels of β-1,4-GalT I and V mRNAs were strong at the crush site, and decreased gradually from crush site to the distal segments. In addition, combined in situ hybridization for β1,4-GalT I and V mRNAs and immunohistochemistry for S100 showed that β1,4-GalT I and V mRNAs were mainly located in Schwann cells. Lectin blot showed that the expression of Galβ1,4GlcNAc group increased at 6 h immediately, reached a peak at 12 h and remained elevated up to 4 w after sciatic nerve crush. In conclusion, β1,4-GalT I and V might play important roles in the regeneration of the injuried sciatic nerve, and upregulation of Galβ1,4GlcNAc group might be correlated with the process of the sciatic nerve injury.     

The theoretical 3D structure of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Cry5Ba

Cry5Ba is a δ-endotoxin produced by Bacillus thuringiensis PS86A1 NRRL B-18900. It is active against nematodes and has great potential for nematode control. Here, we predict the first theoretical model of the three-dimensional (3D) structure of a Cry5Ba toxin by homology modeling on the structure of the Cry1Aa toxin, which is specific to Lepidopteran insects. Cry5Ba resembles the previously reported Cry1Aa toxin structure in that they share a common 3D structure with three domains, but there are some distinctions, with the main differences being located in the loops of domain I. Cry5Ba exhibits a changeable extending conformation structure, and this special structure may also be involved in pore-forming and specificity determination. A fuller understanding of the 3D structure will be helpful in the design of mutagenesis experiments aimed at improving toxicity, and lead to a deep understanding of the mechanism of action of nematicidal toxins.     

Autoantibody profiling of Chinese patients with autoimmune hepatitis using immunoproteomic analysis

In the present study, immunoproteomic analysis was utilized to systemically characterize global autoantibody profiles in autoimmune hepatitis (AIH). Sera from 21 patients with AIH and 15 healthy controls were analyzed for the antibody reactivity against the protein antigens of HepG2, a human hepatoma cell line. The lysates of HepG2 cells were separated by two-dimensional electrophoresis and then immunoblotted with each serum sample. Matrix-assisted laser desorption/ionization mass spectrometry or/and nanoelectrospray ionization MS/MS were then used to identify antigens, among which a bifunctional enzyme in mitochondrial, fumarate hydratase (FH), was further analyzed by ELISA using recombinant FH as a coating antigen. A total of 18 immunoreactive spots were identified as 13 proteins, 8 of which have not been reported in AIH. Immune reactivity to FH was detected in 66.67% of patients with AIH, 19.35% of patients with primary biliary cirrhosis (PBC), 12.31% of patients with chronic hepatitis B (CHB), 6.35% of patients with chronic hepatitis C (CHC), 11.32% of patients with systemic lupus erythematosus (SLE), and 3.57% of normal individuals. The differences of prevalence between AIH patients and healthy controls as well as other diseases were of statistical significance (P<0.001). These data demonstrate the serological heterogeneity in AIH and suggest the diversity of the mechanisms underlying AIH. FH, recognized mainly in AIH rather than in viral hepatitis and other autoimmune diseases, may have utility in improved diagnosis of AIH.     

Role of gap junction, hemichannels, and connexin 43 in mineralizing in response to intermittent and continuous application of parathyroid hormone

Intermittent administration stimulates bone formation, whereas sustained elevation of parathyroid hormone (PTH) as in hyperparathyroidism stimulates bone resorption. Even though PTH(1-34) is the only anabolic agent clinically approved for the treatment of osteoporosis, the molecular mechanism whereby PTH mediates these opposing effects depending on timing of administration is not well understood. In this study, we sought to determine the involvement of gap junctions and hemichannels, and the protein that forms them, connexin 43 (Cx43), in the effect of PTH(1-34) on osteoblast mineralization. The osteoblast-like cell line MLO-A5 that rapidly mineralizes in culture was used. Intermittent PTH enhances mineralization, whereas continuous PTH inhibits this process. The mineralization was significantly inhibited by 18 beta-glycyrrhetinic acid, an inhibitor known to block gap junctions and hemichannels. When the cells were treated with PTH(1-34), gap junctional coupling was increased; however, the degree of stimulation was similar between intermittent and continuous treatment. The permeabilization to dye was not detected under various intermittent or continuous PTH treatments. On the other hand, the overall level of Cx43 protein increased in response to continuous PTH treatment. In contrast, when the cells were subjected to intermittent treatment overall level of Cx43 was unchanged, but there was an increase of connexons associated with an increase in Cx43 expression on the cell surface. Our results suggest that Cx43 overall expression, connexon formation and cell surface expression are differentially regulated by intermittent and continuous PTH(1-34), implying the involvement of Cx43 and Cx43-forming channels in mediating the effects of PTH on bone formation.     

Raf activation is regulated by tyrosine 510 phosphorylation in Drosophila

The proto-oncoprotein Raf is pivotal for mitogen-activated protein kinase (MAPK) signaling, and its aberrant activation has been implicated in multiple human cancers. However, the precise molecular mechanism of Raf activation, especially for B-Raf, remains unresolved. By genetic and biochemical studies, we demonstrate that phosphorylation of tyrosine 510 is essential for activation of Drosophila Raf (Draf), which is an ortholog of mammalian B-Raf. Y510 of Draf is phosphorylated by the c-src homolog Src64B. Acidic substitution of Y510 promotes and phenylalanine substitution impairs Draf activation without affecting its enzymatic activity, suggesting that Y510 plays a purely regulatory role. We further show that Y510 regulates Draf activation by affecting the autoinhibitory interaction between the N- and C-terminal fragments of the protein. Finally, we show that Src64B is required for Draf activation in several developmental processes. Together, these results suggest a novel mechanism of Raf activation via Src-mediated tyrosine phosphorylation. Since Y510 is a conserved residue in the kinase domain of all Raf proteins, this mechanism is likely evolutionarily conserved.     

Sesquiterpene Glucosides from Chloranthus japonicus Sieb

A new lindenane sesquiterpene glucoside named yinxiancaoside A (1), a new, rare bidesmosidic megastigmane sesquiterpene glucoside named yinxiancaoside B (5), and three known sesquiterpene glucosides, chloranoside A (2), pisumionoside (3), and sarcaglaboside A (4), were isolated from the whole plant of Chloranthus japonicus Sieb. The structures of the new compounds were established by an extensive study of their spectral data, especially 1D- and 2D-NMR. The cytotoxic activity of the isolated compounds against human hepatoma (Hepg-2), human ovarian carcinoma (OV420), and human breast cancer (MCF-7) cells was investigated.     

Computational studies of the binding site of α<Subscript>1A</Subscript>-adrenoceptor antagonists

Aimed at achieving a good understanding of the 3-dimensional structures of human α_1A-adrenoceptor (α_1A-AR), we have successfully developed its homology model based on the crystal structure of β₂-AR. Subsequent structural refinements were performed to mimic the receptor’s natural membrane environment by using molecular mechanics (MM) and molecular dynamics (MD) simulations in the GBSW implicit membrane model. Through molecular docking and further simulations, possible binding modes of subtype-selective α_1A-AR antagonists, Silodosin, RWJ-69736 and (+)SNAP-7915, were examined. Results of the modeling and docking studies are qualitatively consistent with available experimental data from mutagenesis studies. The homology model built should be very useful for designing more potent subtype-selective α_1A-AR antagonists and for guiding further mutagenesis studies. Figure The superposition of β₂-AR crystal structure (gold ribbons) and α_1A-AR homology model (blue ribbons)