Large numbers of interleukins‐22‐ and ‐17A‐producing T helper cells in cholangiocarcinoma related to liver fluke infection

Cholangiocarcinoma (CCA) associated with liver fluke infection involves inflammatory and immune processes; however, whether these involve the proinflammatory cytokine IL‐17A and proliferative cytokine IL‐22 remains unclear. Here, numbers of IL‐22‐ and IL‐17A‐producing Th cells and cytokine concentrations in 30 patients with CCA and long‐term liver fluke infection, 40 patients with liver‐fluke infection but not CCA, and 16 healthy controls were compared. Analyses were performed using immunohistochemistry, flow cytometry, ELISA and RT‐PCR. Immunohistochemical staining showed weaker expression of IL‐22 and IL‐17A in patients with CCA with than in those without liver fluke infection (P < 0.01). Flow cytometry revealed significantly greater median proportions of IL‐22‐producing T helper cells in patients with CCA (2.2%) than in those without it (0.69%) or controls (0.4%, P < 0.001). Similar results were obtained for IL‐17A‐producing T helper cells. ELISA revealed plasma concentrations of IL‐22 were 1.3‐fold higher in patients with CCA than in those without it and 4.6‐fold higher than in controls (P < 0.001). Plasma concentrations of IL‐17A were 2.5‐fold higher in patients with CCA than in those without it, and 21‐fold higher than in controls (P < 0.001). Amounts of IL‐22 and IL‐17A mRNAs in blood were significantly higher in patients with CCA than in the other two groups. Proportions of CD4⁺CD45RO⁺ T cells producing IL‐22 correlated with proportions producing IL‐17A (r = 0.759; P < 0.001), and plasma concentrations of IL‐22 correlated with those of IL‐17A (r = 0.726; P < 0.001). These results suggest that both IL‐17A and IL‐22 affect development of CCA related to liver fluke infection.

     

Activation of NFKB-JMJD3 signaling promotes bladder fibrosis via boosting bladder smooth muscle cell proliferation and collagen accumulation

Chronic cystitis is characterized by the hyperplasia and fibrosis of the bladder wall as well as attenuated compliance of the bladder. To further unravel its underlying molecular mechanism, the role of NFκB-JMJD3 signaling pathway in cystitis induced bladder fibrosis was investigated. Jmjd3 and Col1/3 expression was detected in a cystitis mouse model that was developed by intraperitoneal injection of cyclophosphamide (CYP). Human bladder smooth muscle cells (hBSMCs) were stimulated in vitro with lipopolysaccharide (LPS), and the cell proliferation and collagen accumulation were detected using EdU, CCK8, flow cytometry, qPCR, western blotting and immunofluorescence assays. Furthermore, the effects of NFκB and JMJD3 on cell proliferation and collagen accumulation were investigated using its selective antagonists, JSH23 and GSK-J4, respectively. CYP induced cystitis significantly increased Jmjd3, Col1 and Col3 expression in the bladder muscle cells. Furthermore, LPS stimulation markedly activated NFκB signaling and elevated JMJD3 expression in hBSMCs, and the activation of NFκB-JMJD3 signaling significantly promoted cell proliferation and collagen accumulation by upregulating CCND1 and COL1/3 expression, respectively. Our study reveals the critical role of NFκB-JMJD3 signaling in cystitis induced bladder reconstruction by regulating hBSMC proliferation and extracellular matrix (ECM) deposition, and these findings provide an avenue for effective treatment of patients with cystitis.     

不同耐性水稻幼苗根系对镉胁迫的形态及生理响应

采用水培试验,以两个耐镉性不同的水稻品种为材料,研究了不同浓度镉胁迫对水稻幼苗根系形态、根系活力、游离脯氨酸含量及抗氧化酶活性的影响。结果表明:低于5 μmol/L Cd胁迫对2个水稻品种总根长、根表面积、根体积、根干重、根系活力无明显影响,在1 μmol/L Cd时,甚至起促进作用。随Cd浓度增加表现出一定的抑制效应,秀水63在10 μmol/L Cd胁迫下根系形态、根系活力明显受到抑制,而秀水09在25 μmol/L Cd胁迫下明显受抑。随Cd胁迫浓度的增加,游离脯氨酸含量、丙二醛(MDA)含量、超氧化物歧化酶(SOD)、过氧化物酶(POD)活性均呈上升趋势,两品种相比,秀水09的游离脯氨酸含量、SOD和POD增幅大于秀水63,而MDA含量增幅小于秀水63;过氧化氢酶(CAT)活性变化表现为先上升后下降,10-100 μmol/L Cd胁迫下秀水63根系中CAT活性明显低于秀水09。总之,水稻对Cd毒害响应存在明显的品种差异,且Cd胁迫下根系生理响应的差异是品种间耐性差异的重要原因之一。     

Enzymatic activity of glucose oxidase covalently wired via viologen to electrically conductive polypyrrole films

The surface functionalization of an electrically conductive polypyrrole film (PPY) with a viologen, (N-(2-carboxyl-ethyl)-N'-(4-vinyl-benzyl)-4,4'-bipyridinium dichloride, or CVV) for the covalent immobilization of glucose oxidase (GOD) has been carried out. The viologen was first synthesized and graft polymerized on PPY film. It then served as an anchor via its carboxyl groups for the covalent immobilization of GOD. The surface composition of the as-functionalized substrates was characterized by X-ray photoelectron spectroscopy (XPS). The effects of the CVV monomer concentration on the CVV-graft polymer concentration and the amount of GOD immobilized on the surface were investigated. The activity of the immobilized GOD was compared with that of free GOD and the kinetic effects were also obtained. The cyclic voltammetric (CV) response of the GOD-functionalized PPY substrates was studied in a phosphate buffer solution under an argon atmosphere. The CV results support the mechanism in which CVV acts as a mediator to transfer electron between the electrode and enzyme, and hence regenerating the enzyme in the enzymatic reaction with glucose. High sensitivity and linear response of the enzyme electrode was observed with glucose concentration ranging from 0 to 20 mM.     

Long-term production of soluble human Fas ligand through immobilization of <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis> in a fibrous bed bioreactor

The production of recombinant glycoproteins in Dictyostelium discoideum by conventional cell culture methods was limited by low cell density as well as low growth rate. In this work, cotton towel with a good adsorption capability for D. discoideum cells was used as the immobilization matrix in an external fibrous bed bioreactor (FBB) system. With batch cultures in the FBB, the concentration of immobilized cells in the cotton fiber carrier increased to 1.37 × 10⁸ cells per milliliter after 110-h cultivation, which was about tenfold higher than the maximal cell density in the conventional free-cell culture. Correspondingly, a high concentration of soluble human Fas ligand (hFasL; 173.7 μg l⁻¹) was achieved with a high productivity (23 μg l⁻¹ h⁻¹). The FBB system also maintained a high density of viable cells for hFasL production during repeated-batch cultures, achieving a productivity of 9∼10 μg l⁻¹ h⁻¹ in all three batches studied during 15 days. The repeated-batch culture using immobilized cells of D. discoideum in the FBB system thus provides a good method for long-term and high-level production of hFasL.     

Enhancement of 5-aminolevulinate production with recombinant Escherichia coli using batch and fed-batch culture system

5-Aminolevulinate (ALA) production with recombinant Escherichia coli Rosetta (DE3)/pET28a(+)-hemA was studied. In batch fermentation, the addition of glucose and glycine was effective to improve ALA production. Then the fed-batch fermentation was conducted with continuous feeding of precursors. When the concentrations of succinic acid and glycine were 7.0 g/l and 4.0 g/l, respectively, in the feeding, the ALA yield reached 4.1g/l. But the molar yield (ALA/glycine) was decreased in the fed-batch fermentation compared to batch fermentation. And it was found that the pH control during fed-batch cultivation was very important for the cell growth and ALA production. A two-stage pH value controlling strategy was suggested, in which, the pH value in the first 6h was regulated at pH 5.9, after then at pH 6.2, and the ALA yield was as high as 6.6g/l via fed-batch fermentation.     

Simultaneous determination of dronedarone and its active metabolite debutyldronedarone in human plasma by liquid chromatography-tandem mass spectrometry: application to a pharmacokinetic study

Dronedarone is a derivative of amiodarone--a popular antiarrhythmic drug. It was developed to overcome the limiting iodine-associated toxicities of amiodarone. Debutyldronedarone is a major circulating active metabolite of dronedarone in humans. To investigate the pharmacokinetics of dronedarone, a rapid, simple, and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to simultaneously determine dronedarone and debutyldronedarone in human plasma using amiodarone as internal standard (IS). Acetonitrile with IS was used to precipitate proteins from a 50-μL aliquot of plasma. Effective chromatographic separation was performed on a CAPCELL PAK C(18) MG (100 mm × 4.6 mm, 5 μm) column with gradient elution (5 mmol/L ammonium acetate-acetonitrile, with each phase containing 0.2% acetic acid) at a flow rate of 0.7 mL/min. Complete separation was achieved within 5.5 min. Detection was carried out on an tandem mass spectrometer in multiple reaction monitoring mode using a positive atmospheric pressure chemical ionization interface. A lower limit of quantification of 0.200 ng/mL was achieved for both dronedarone and debutyldronedarone, with acceptable precision and accuracy. The linear range of the method was from 0.200 to 200 ng/mL for each analyte. Intra- and inter-day precisions were lower than 7.2% in relation to relative standard deviation, while accuracy was within ±5.1% in terms of relative error for analytes. Our findings demonstrate the successful application of the validated LC-MS/MS method to a pharmacokinetic study after a single oral administration of 400mg dronedarone to six healthy volunteers.     

中国菊属一些种的分支分类学研究

运用分支分析方法研究了12种原产中国的野生菊属植物的系统发育关系,并引入了若干栽培品种及部分杂种一代植株作为分析材料。研究结果表明：分支分析方法有效而准确地将分类群分类,并揭示出毛华菊（Dendranthemarestitum）与菊花（Dendranthema×gran-diflorum）同为菊属植物中进化程度较高的种,部分种间杂种也已进入栽培类群。同时还发现不同性状在各品种间平行进化的现象。根据上述结果,作者讨论了中国菊属植物的系统进化及菊花起源问题。     

Two distinct CCR5 domains can mediate coreceptor usage by human immunodeficiency virus type 1.

The chemokine receptor CCR5 is the major fusion coreceptor for macrophage-tropic strains of human immunodeficiency virus type 1 (HIV-1). To define the structures of CCR5 that can support envelope (Env)-mediated membrane fusion, we analyzed the activity of homologs, chimeras, and mutants of human CCR5 in a sensitive gene reporter cell-cell fusion assay. Simian, but not murine, homologs of CCR5 were fully active as HIV-1 fusion coreceptors. Chimeras between CCR5 and divergent chemokine receptors demonstrated the existence of two distinct regions of CCR5 that could be utilized for Env-mediated fusion, the amino-terminal domain and the extracellular loops. Dual-tropic Env proteins were particularly sensitive to alterations in the CCR5 amino-terminal domain, suggesting that this domain may play a pivotal role in the evolution of coreceptor usage in vivo. We identified individual residues in both functional regions, Asp-11, Lys-197, and Asp-276, that contribute to coreceptor function. Deletion of a highly conserved cytoplasmic motif rendered CCR5 incapable of signaling but did not abrogate its ability to function as a coreceptor, implying the independence of fusion and G-protein-mediated chemokine receptor signaling. Finally, we developed a novel monoclonal antibody to CCR5 to assist in future studies of CCR5 expression.