Facile Fluorescence‐Based Detection of PAD4‐Mediated Citrullination

The post‐translational modifications of histone proteins are highly diverse and dynamic processes. It is becoming increasingly evident that modifying histone proteins can have a direct influence on both cellular homeostasis and disease states. Protein arginine deiminase 4 (PAD4) is an enzyme that converts peptidyl‐arginine to citrulline. The overexpression of PAD4 has been found in numerous types of human cancer and autoimmune diseases. We report a new, facile, fluorescence‐based assay for the detection of PAD4 activity that exploits the substrate specificity of trypsin to monitor the citrullination reaction carried out by PAD4 based on the fact that, upon citrullination, the positively charged arginine side chain is converted to the neutral citrulline. We show that the assay can be performed rapidly with readily available reagents and that it responds accordingly to a known PAD4 inhibitor.     

Isolation and characterisation of a kallikrein-like enzyme from Agkistrodon halys pallas snake venom

BACKGROUND: Viper snake venoms contain a great variety of toxic proteins. These components mediate their toxicity by either stimulating or inhibiting the haemostatic system of human victims or experimental animals, resulting in common clinical complications of blood clotting or uncontrolled haemorrhage. Therefore it is deemed important to isolate the active component(s) from snake venom with kallikrein‐like activity. RESULTS: A kallikrein‐like proteinase of Agkistrodon halys pallas snake venom, designated AHP‐Ka, was purified by anion exchange chromatography and affinity chromatography. Physicochemical studies showed that the purified enzyme was a 34 kDa monomeric glycoprotein, the molecular weight of which decreased to 26 kDa after deglycosylation with peptide N‐glycosidase F (PNGase F). Sequence studies on the NH₂‐terminal region of the protein indicated that AHP‐Ka shared a high degree of sequence homology with other serine proteinases from snake venoms. AHP‐Ka showed high catalytic activity and kallikrein‐like activity on substrates such as arginine esterase BAEE and chromogenic H‐D‐Pro‐Phe‐Arg‐pNA·2HCl (S‐2302) and was inhibited by protease inhibitor phenylmethylsulfonyl fluoride (PMSF). CONCLUSION: The results showed that AHP‐Ka isolated from A. halys pallas snake venom and purified by anion exchange chromatography and affinity chromatography is in fact a kallikrein‐like enzyme. Copyright © 2011 Society of Chemical Industry     

Induction of mud crab (Scylla paramamosain) tropomyosin and arginine kinase specific hypersensitivity in BALB/c mice

BACKGROUND: Shellfish hypersensitivity is among the most common food allergies. The allergens tropomyosin (TM) and arginine kinase (AK) from mud crab (Scylla paramamosain) were purified to homogeneity. BALB/c female mice were sensitized with TM and AK by intragastric administration. Mice treated with normal saline served as the negative control (NC) group. RESULTS: Compared with NC group, mice that were treated with TM and AK developed reduced activity; meanwhile, their scratching behavior and specific‐IgE level were increased. Specific‐CD4 + T cells were significantly elevated in the splenocyte cultures of the mice upon TM and AK stimulation. However, compared with the positive control group (ovalbumin, OVA), there was no significant difference. The expression of IL‐4 in culture cells stimulated by TM, AK, and OVA group showed significant differences from the NC group, respectively. CONCLUSION: These results indicated that a BALB/c mouse model for sensitization to TM and AK from mud crab was successfully established, and the Th2 response was observed, displaying increased immunoglobulin E levels, together with the production of interleukin 4 and allergic symptoms. Copyright © 2011 Society of Chemical Industry     

偶联剂对蒙脱石吸附精氨酸的影响

作者从研究精氨酸吸附曲线出发,分别利用戊二醛(GA)、γ-甲基丙烯酰氧基丙基三甲氧基硅垸(KH570)处理蒙脱石,研究了不同处理方式下蒙脱石对精氨酸的吸附作用.结果显示,精氨酸吸附进入蒙脱石是静电作用、疏水作用和氢键等共同作用的结果,偶联剂存在的情况下会有部分共价键生成.XRD和FT-IR显示KH570使MMT表面结构发生改变,而GA则没有明显改变MMT的结构,但GA的使用可能更容易导致化学键的生成.偶联剂的使用可以增加吸附量,但对层间距影响不明显.该项研究对于蒙脱石的有机改性及药物负载等方面的应用研究具有重要意义.     

发酵酒精饮品中氨基甲酸乙酯前体物的代谢调控机制研究进展

氨基甲酸乙酯（ethyl carbamate,EC）又称脲烷,是一种广泛存在于发酵酒精饮品中的氨基甲酰类物质。由于EC目前被证实能对实验动物造成多位点致癌,并且对人类具有潜在致癌性,因此其对发酵食品安全的影响是目前发酵工业中面临的重要问题。EC主要由氨基甲酰类物质和乙醇自发作用形成,其中重要的前体物包括尿素和瓜氨酸,并且二者均来源于精氨酸的分解代谢,因此对这三者代谢调控机制的研究能够为EC抑制途径的探索提供新的思路,本文主要就尿素、瓜氨酸及精氨酸分别在酿酒酵母及乳酸菌中的代谢调控机制进行系统的概述,并对未来EC的相关研究进行展望。     

大蒜精油对熏马肠中德氏乳杆菌产腐胺的影响机制

研究不同质量浓度大蒜精油对熏马肠中德氏乳杆菌产腐胺的影响机制。利用反转录实时荧光定量聚合酶链式反应技术分析供试菌在不同大蒜精油质量浓度下的胍基丁胺脱亚胺酶相关基因表达情况,使用超高效液相色谱连续48 h监测纯菌体系中的腐胺产量;并将德氏乳杆菌接入到含不同质量浓度大蒜精油的熏马肠中发酵28 d,期间取样测其菌落总数、pH值及腐胺积累量。结果表明：无论是在纯菌培养还是在熏马肠发酵过程中,大蒜精油都能明显抑制细菌的增长,在纯菌体系中会明显抑制pH值的下降,但对熏马肠的最终pH值影响不明显;大蒜精油对调节基因aguR的转录影响不明显,但能极显著抑制操纵子aguBDAC的转录（P＜0.01）;增加纯菌体系以及熏马肠发酵过程中的大蒜精油质量浓度均能明显减少腐胺的生成。     

Dietary Plant Sterols Supplementation Increases In Vivo Nitrite and Nitrate Production in Healthy Adults: A Randomized,Controlled Study

A randomized, double‐blinded, placebo‐controlled and crossover study was conducted to simultaneously measure the effects, 3 h after consumption and after 4‐wk daily exposure to plant sterols‐enriched food product, on in vivo nitrite and nitrate production in healthy adults. Eighteen healthy participants (67% female, 35.3 [mean] ± 9.5 [SD] years, mean body mass index 22.8 kg/m²) received 2 soy milk (20 g) treatments daily: placebo and one containing 2.0 g free plant sterols equivalent of their palmityl esters (β‐sitosterol, 55%; campesterol, 29%; and stigmasterol, 23%). Nitrite and nitrate concentrations were measured in the blood plasma and urine, using stable isotope‐labeled gas chromatography‐mass spectrometry. L‐arginine and asymmetric dimethylarginine concentrations in blood serum were measured using commercially available enzyme immunoassays. Nitrite and nitrate concentrations in blood plasma (nitrite 5.83 ± 0.50 vs. 4.52 ± 0.27; nitrate 15.78 ± 0.96 vs. 13.43 ± 0.81 μmol/L) and urine (nitrite 1.12 ± 0.22 vs. 0.92 ± 0.36, nitrate 12.23 ± 1.15 vs. 9.71 ± 2.04 μmol/L) were significantly elevated after 4‐wk plant sterols supplementation Placebo and 3‐h treatments did not affect the blood plasma and urinary concentrations of nitrite and nitrate. Circulating levels of L‐arginine and asymmetric dimethylarginine were unchanged in the placebo and treatment arms. Total plant sterols, β‐Sitosterol, campesterol, and stigmasterol concentrations were significantly elevated after 4‐wk treatments compared to the placebo and 3‐h treatments. Blood plasma nitrite and nitrate concentrations correlated significantly with the plasma total and specific plant sterol concentrations. Our results suggest that dietary plant sterols, in the combination used, can upregulate nitrite, and nitrate production in vivo.     

枯草芽孢杆菌精氨酸脱羧酶基因speA的表达与蛋白纯化

根据枯草芽孢杆菌（Bacillus subtilis）BJ3-2的精氨酸脱羧酶（ADC）的编码基因speA序列设计特异性酶切引物,克隆基因speA序列。测序结果显示,基因speA全长为1 473 bp,编码490个氨基酸,分子质量为58 ku。基因speA克隆至原核表达载体,获得重组菌pET28a-speA/BL21,十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）结果显示,1.0 mmol/L的异丙基β-D-硫代半乳糖苷（IPTG）28 ℃诱导4 h,上清液和菌体均能表达出ADC蛋白,上清液经纯化、透析、冷冻干燥可获得纯度97%的ADC酶,酶联免疫吸附检测（ELISA）ADC酶活为16 780 U/mg。为speA基因的表达、纯化及酶学性质研究奠定了理论基础。     

响应面法优化阴离子淀粉微球吸附性能研究

以精氨酸为模型药物,采用响应面法优化阴离子淀粉微球的吸附性能。在单因素试验的基础上选取离子化剂用量、反应温度、浸泡时间和反应时间为影响因子,应用中心组合(CCD)进行四因素五水平的试验设计,以阴离子淀粉微球吸附量作为响应值,进行响应面分析(RSA)。结果表明,阴离子淀粉微球吸附精氨酸含量的最佳优化条件为三聚磷酸钠用量0.82g、反应温度80.52℃、浸泡时间12h、反应时间1h。精氨酸吸附量预测值为3.341mg/g,验证值为3.308mg/g,与预测值相差0.033mg/g,比阴离子化前吸附量提高了95.16%。     

N-octyl-N-arginine-chitosan (OACS) micelles for gambogic acid oral delivery: preparation,characterization and its study on in situ intestinal perfusion

Context: Gambogic acid (GA) can inhibit the growth of various cancer cells. However, the low bioavailability caused by insolubility, limits its clinical application. L-arginine is always used with GA to form a complex to obtain the higher solubility. Moreover, guanidyl group from arginine, which can facilitate the cellular uptake, was identified.

Objective: In this study, L-arginine and chitosan (CS) were used for the first time to prepare N-octyl-N-arginine CS (OACS), a novel amphiphilic carrier for GA with solubility- and absorption-enhancing functions; the characterization of the GA loaded OACS micelles (GA-OACS) and its absorption-enhancing effect were also investigated.

Materials and methods: GA-OACS were prepared by the dialysis method. The formed micelles were characterized and evaluated by atomic force microscope (AFM), dynamic light scattering, differential scanning calorimeter (DSC), solubility test, in vitro release and in situ intestinal perfusion.

Results: The GA-OACS micelles were successfully prepared attaining a 35.3% drug loading and 82.2% entrapment efficiency. GA-OACS had a homogeneous particle size of 160.3?nm; +21.8?mv zeta potential with smooth continuous surface was observed by using AFM. DSC diagram suggested that GA was encapsulated in the micelles. Meanwhile, GA encapsulated in micelles exhibited a desirable slow release in vitro experiment. The solubility of GA in OACS micelles was increased up to 3.16?±?0.13?mg/mL, 2320 times than that of free GA. The single pass perfusion showed that the absorption of GA-OACS micelles was enhanced 3.6-fold, 2.1-fold and 2.2-fold for jejunum, ileum and colon, respectively.

Discussion and conclusion: OACS provided excellent ability of drug loading, increasing solubility and enhanced absorption for GA, which indicated that OACS micelles as an oral drug delivery carrier may have potential research and application values.