Determination of egg and milk allergen in food products by liquid chromatography-tandem mass spectrometry based on signature peptides and isotope-labeled internal standard

The aim of this work was to develop a liquid chromatography-tandem mass spectrometry method for the determination of milk allergen and egg allergen in food products.Signature peptides GGLEPINFQTAADQAR,VGINYWLAHK,VLVLDTDYK,FFVAPFPEVFGK,and NAVPITPTLNR were confirmed and synthesized as the quantitative peptide of ovalbumin,α-lactalbumin,β-lactoglobulin,α_(S1)-casein andα_(S2)-casein,the relative isotope-labeled internal standards were used in the quantitative analysis.Linear range was in the range of0.5-5000.0 nmol/L for egg and milk allergen in bread,cake,cookie,rice crust and wheat flour samples with free from egg and milk,the limits of detection of milk allergens and egg allergen were in the range between0.94 mg/100 g and 56.71 mg/100 g,limits of quantification of milk allergens and egg allergen were in the range between 2.36 mg/100 g and 141.78 mg/100 g.The recoveries ranged from 76.7%to 122.8%,the relative standard deviations were in the range of 1.60%-15.60%.The developed method has been successfully used for the detection of egg and milk allergen in various food samples.

Multifaceted neuroprotective effects of(-)-epigallocatechin-3-gallate(EGCG)in Alzheimer's disease:an overview of pre-clinical studies focused onβ-amyloid peptide

Alzheimer’s disease(AD)is the most common neurodegenerative disease characterized by cognitive decline and memory impairment.Many lines of evidence indicate that excessiveβ-amyloid peptide(Aβ)generation and aggregation play pivotal roles in the initiation of AD,leading to various biochemical alteration including oxidative damage,mitochondrial dysfunction,neuroinflammation,signaling pathway and finally resulting in neuronal death.AD has a complex pathogenic mechanism,and a single-target approach for anti-AD strategy is thus full of challenges.To overcome these limitations,the present study focused to review on one of multiple target-compounds,(-)-epigallocatechin-3-gallate(EGCG)for the prevention and treatment of AD.EGCG is a main bioactive polyphenol in green tea and has been reported to exert potent neuroprotective properties in a wide array of both cellular and animal models in AD.This review demonstrated multiple neuroprotective efficacies of EGCG by focusing on the involvement of Aβ-evoked damage and its Aβregulation.Furthermore,to understand its mechanism of action on the brain,the permeability of the blood-brain barrier was also discussed.

Protective effects of proanthocyanidins on beta-amyloid peptide (25-35)-induced PC12 cell apoptosis by blocking S-phase and increasing p53 gene expression

BACKGROUND： Current studies related to the effects of proanthocyanidins on Alzheimer＇s disease have focused primarily on the signal transduction pathway of cellular apoptosis. However, the influence of p53 gene expression on cell cycle regulation, with regard to the protective mechanisms of proanthocyanidins, has not been reported. OBJECTIVE： To observe the effect of proanthocyanidins on cell cycle distribution, cellular apoptosis and p53 gene expression in β-amyloid peptide （25-35） （Aβ25-35）-induced PC12 cells cultured in serum-free media, and to investigate the molecular neuroprotective mechanisms of proanthocyanidins with regard to cell cycle regulation. DESIGN, TIME AND SETTING： A parallel, controlled, at the Institute of Biochemistry and Molecular Biology cellular, and molecular study was performed Guangdong Medical College from July 2006 to July 2008. MATERIALS： Proanthocyanidins were provided by Nanjing Xuezi Medical and Chemical Research Center, China; Aβ25-35 was provided by Sigma, USA; PC12 cells were provided by the Institute of Basic Medical Science, Academy of Military Medical Sciences; and rabbit anti-p53 polyclonal antibody was provided by Santa Cruz Biotechnology, USA. METHODS： PC12 cells were cultured in serum-free media for 24 hours. Cells from the model group were treated with 25 μmol/L Aβ25-35 for 24 hours. Cells in the drug protection group were pre-treated with 30 mg/L proanthocyanidins for 1 hour and then treated with 25 μmol/LAβ2^-35 for 24 hours. The control group was not treated. MAIN OUTCOME MEASURES： Flow cytometry was used to detect cell cycle distribution and rate of apoptosis; reverse-transcriptase polymerase chain reaction was used to detect p53 mRNA expression; and Western blot was used to detect p53 protein expression. RESULTS： After treating with 25 μmol/LAβ25-35 for 24 hours, the rate of apoptosis and the percentage of cells in S phase were significantly increased （P 〈 0.01 ）, and p53 mRNA and protein expressions were decreased. Pretreatment with proanthocyanidins for 1 hour blocked the increase in apoptosis and the percentage of cells in S phase in Aβ25-35-induced PC12 cells （P 〈 0.01 ） and increased p53 mRNA and protein expressions. CONCLUSION： Proanthocyanidins blocked apoptosis and S-phase arrest in Aβ25-35-induced PC12 cells cultured in serum-free media. The protective mechanism could be related to increased p53 mRNA and protein expressions.

Hydrogen sulfide inhibits beta-amyloid peptide-induced apoptosis in PC12 cells and the underlying mechanisms

BACKGROUND：Studies have demonstrated that hydrogen sulfide（H2S） levels are 55% lower in brains of Alzheimer＇s disease（AD） patients than in age-matched normal individuals,which suggests that H2S might be involved in some aspects of AD pathogenesis.OBJECTIVE：To observe the protective mechanisms of varied concentrations of H2S against β-amyloid-peptide（Aβ） induced apoptosis in pheochromoytoma（PC12） cells,and to analyze the pathway of action.DESIGN,TIME AND SETTING：A controlled,observational,in vitro experiment was performed at Neurophysiology Laboratory in Zhongshan Medical School,Sun Yat-sen University between July 2006 and May 2007.MATERIALS：PC12 cells were provided by the Animal Experimental Center of Medical School of Sun Yat-sen University.Glybenclamide,rhodamine123,and dihydrorhodamine123 were purchased from Sigma（USA）.METHODS：PC12 cells were incubated at 37 ℃ in a 5% CO2-enriched incubator with RPMI-1640 medium,supplemented with 5% horse-serum and 10% fetal bovine serum.Cells in logarithmic growth curves received different treatment：The PC12 cells were maintains at 37 ℃ with the original medium,then incubated in Aβ25-35,sodium hydrosulfide（NaHS）,glybenclamide,NaHS＋ Aβ25-35,or pretreated with glybenclamide 30 minutes prior to administration of and Aβ25-35,respectively.MAIN OUTCOME MEASURES：（1） The survival rate of PC12 cells was detected by MTT assay and Hoechst staining.（2） The apoptosis rate of PC12 cells was detected utilizing flow cytometry with propidium iodide staining,and morphological changes of apoptotic cells were observed.（3） The mitochondrial membrane potential was detected by Rhodamine123-combined flow cytometry.（4） The intracellular reactive oxygen species content was detected by dihydrorhodamine123-combined flow cytometry.RESULTS：Aβ25-35 induced significantly decreased viability and increased percentage of apoptosis in PC12 cells,as well as dissipated mitochondrial membrane potential expression and an overproduction of reactive oxygen species.When PC12 cells were co-treated with NaHS and Aβ25-35,the decreased cell viability induced by 20 μmol/L Aβ25-35 was concentration-dependently blocked by NaHS（50,100,and 200 μmol/L）.NaHS（100 μmol/L） obviously reduced the percentage of apoptotic PC12 cells induced by 20 μmol/L Aβ25-35.In addition,100 μmol/L NaHS inhibited mitochondrial membrane potential dissipation and reactive oxygen species overproduction.When the ATP-sensitive K channel（KATP） inhibitor,glybenclamide,was administered 30 minutes prior to NaHS and Aβ25-35 treatment,the NaHS-dependent cellular protection was partly blocked.This resulted in reduced PC12 cell viability and increased the percentage of apoptosis,as well as significantly blocked mitochondrial membrane potential preservation and inhibited reactive oxygen species overproduction due to NaHS treatment.CONCLUSION：NaHS protected PC12 cells against Aβ25-35-induced damage.NaHS-dependent cellular protection was associated with mitochondrial membrane potential preservation and inhibition of reactive oxygen species overproduction.The KATP channel inhibitor,glybenclamide,significantly blocked the cellular protective effects of NaHS,indicating that KATP channel activation plays an important role in NaHS-induced protection of PC12 cells to Aβ25-35-induced damage.

Effects of Yizhi Capsule (益智胶囊) on Learning and Memory Disorder and β-amyloid Peptide Induced Neurotoxicity in Rats

To explore the effects of Yizhi Capsule （益智胶囊, YZC） on learning and memory disorder and β-amyloid peptide induced neurotoxicity in rats. Methods： Various doses of YZC were administered to Sprague-Dawley （SD） rats for 8 consecutive days, twice a day. On the 8th day of the experiment, scopolamine hydrobromide was intraperitoneally injected to every rat and Morris water maze test and shuttle dark avoidance test were carried out respectively to explore the changes of learning and memory capacities in the rats. Resides, after the cerebral cortical neurons of newborn SD rats aged within 3 days were cultured in vitro for 7 days, drug serum containing YZC was added to the cultured neurons before or after β amyloid peptide25-35 （Aβ25-35） intoxication to observe the protective effect of YZC on neurotoxicity by MTT assay and to determine the LDH content in the supernatant. Results： Compared with those untreated with YZC, the rats having received YZC treatment got superiority in shorter time of platform seeking in Morris water maze test, as well as elongated latent period and less times of error in shuttle dark avoidance test. On the cultured neurons, YZC drug serum could effectively increase the survival rate of Aβ25-35 intoxicated neurons and reduce the LDH contents in cultured supernatant. Conclusion： YZC has an action of improving learning and memory disorder, and good protective effect on Aβ25-35 induced neurotoxicity in SD rats. KEY WORDS

STUDY ON THE THERAPEUTIC EFFECTS OF GINSENOSIDE Rg-1 AND GASTRODINE ON AD MODEL RATS INDUCED BY β-AMYLOID PEPTIDE (25-35)

Objective To study the therapeutic effects of Ginsenoside Rg-1 and Gastrodine on rats model of Alzheimer's disease(AD). Methods Aggregated β-Amyloid peptide (25-35) was injected into the lateral ventricle of rats to establish AD models. Ginsenoside Rg-1, Gastrodine and Ginsenoside Rg-1+Gastrodine were intraperitoneally injected into rats of each test group(Ginsenoside Rg-1∶10mg/kg·day; Gastrodine 100mg/kg·day) for 4 weeks, the rats of control group received equal volume of saline. Passive avoidance task and Morris maze test were done to assess the ability of learning and memory. The content of superoxide dismutase (SOD), malondiadehyde (MDA), total-antioxidative capability (T-AOC), Choline acetyltransferase (ChAT) and acetylcholinesterase (AchE) in brain tissue were measured. Results Ginsenoside Rg-1 and Gastrodine significantly improved learning and memory deficits in the rats with AD induced by β-Amyloid peptide (25-35) (P<0.05). Ginsenoside Rg-1+Gastrodine group were better than Ginsenoside Rg-1 group and Gastrodine group (P<0.05). Ginsenoside Rg-1 reduced the increase of SOD, MDA, but inhibited the decrease of T-AOC, AchE and ChAT; Gastrodine reduced the increase of SOD, MDA, while inhibited the decrease of T-AOC. Gastrodine could also prevent the activity of ChAT and AchE decline in AD rats. Conclusion Both Ginsenoside Rg-1 and Gastrodine have therapeutic effects on rats with AD; Ginsenoside Rg-1 and Gastrodine injection at the same time were better than only using one of them. Their mechanisms might different. Ginsenoside Rg-1 can not only inhibit peroxidation but also increase the activity of AchE and ChAT in brain tissue, while Gastrodine can inhibit peroxidation only, but it can't prevent the decline of ChAT and AchE activity in AD rats.

一种法氏囊源十一肽对小鼠短期喂养毒性试验

为了研究一种法氏囊源十一肽对小鼠临床症状、内脏器官发育、生长性能、血常规及血清生化指标等的影响,试验采用28 d短期喂养试验,将72只健康ICR系小鼠随机分为4组,分别为高、中、低剂量组及空白对照组,每组3个重复,每个重复6只(雌雄各半),高、中、低剂量组分别按体重灌胃1000,100,10 mg/kg的法氏囊源十一肽溶液(0.2 mL/只),空白对照组灌胃等量纯化水,观察小鼠临床症状,试验结束后统计生长性能指标,采集小鼠的肠、肝脏、脾脏、肾脏、心脏、睾丸及卵巢组织进行病理组织学检查,计算脏器系数,检测血常规及血清生化指标。结果表明:各组小鼠的眼部、鼻孔、被毛、雄鼠阴部、雌鼠乳房及阴部均未出现异常变化;高、中、低剂量组小鼠内脏器官未见病理组织学变化,与空白对照组均呈健康状态;高、中、低剂量组小鼠的生长性能指标、脏器系数、血常规及血清生化指标与空白对照组比较均差异不显著(P>0.05)。说明28 d短期灌胃法氏囊源十一肽对小鼠无毒性作用。

补肾化痰汤联合来曲唑治疗多囊卵巢综合征临床研究

目的:探讨补肾化痰汤联合来曲唑治疗多囊卵巢综合征的效果及对患者成纤维细胞生长因子21(FGF-21)、生长激素释放肽(Ghrelin)及内分泌的影响。方法:选择200例多囊卵巢综合征患者,采用随机数字表法分为试验组和对照组,各100例。对照组给予来曲唑治疗,试验组在对照组的基础上给予补肾化痰汤治疗。比较两组临床疗效、FGF-21、Ghrelin、内分泌、卵巢功能变化情况及不良反应发生情况。结果:治疗后,两组总有效率比较差异有统计学意义(P<0.05);治疗后,两组FGF-21水平均降低,试验组降低更为明显,Ghrelin水平均升高,试验组升高更为明显(P<0.05);治疗后,两组促黄体生成素(LH)水平均降低,试验组降低更为明显,卵泡雌激素(FSH)、雌二醇(E2)水平均升高,试验组升高更为明显(P<0.05);治疗后,两组卵巢体积、卵泡个数及阻力指数水平均降低,试验组降低更为明显,搏动指数水平均升高,试验组升高更为明显(P<0.05);治疗期间,不良反应主要有恶心呕吐、腹部不适及月经量增多,两组发生率比较差异无统计学意义(P>0.05)。结论:在多囊卵巢综合征中应用补肾化痰汤联合来曲唑效果显著,可能与其可有效改善患者FGF-21、Ghrelin及内分泌水平有关。

Cholesterol Depletion Reduces the Internalization of β-Amyloid Peptide in SH-SY5Y Cells

Deposition of amyloid in the brain is a critical step in the pathogenesis of Alzheimer＇s disease. The endocytosis of β-amyloid peptide （Aβ） is an important factor among the many factors that contribute to the genesis of amyloid deposits. Since cholesterol participates in many important physiological processes, the present work investigated the relationship between the cellular cholesterol content and the endocytosis of the exogenic Aβ, and found that reduction of the cholesterol content by methyl-β-cyclodextrin could reduce the endocytosis of AI3. The study indicates that the endocytosis of Aβ is partly mediated by cholesterol.

蛋清肽和姜黄素协同增强多糖基高内相Pickering乳液的界面结构实现活性保护

食品级固体颗粒稳定的高内相Pickering乳液(high internal phase Pickering emulsion,HIPPEs)具有优异的生物安全性、结构稳定性,且具有递送功能因子的潜力,可用作食品油墨开发个性化、营养丰富的高值化产品。基于β-环糊精(β-cyclodextrin,β-CD)与壳聚糖盐酸盐(chitosan hydrochloride,CHC)相互作用以稳定多糖基HIPPEs。结果表明,当CHC添加量为0.4%、水相pH值为4.0时,在静电斥力和空间位阻的共同作用下,β-CD和CHC形成致密的交联互穿网络,稳定的HIPPEs液滴大小均匀,粒径最小,为(26.35±8.83)nm。该HIPPEs可实现对以蛋清肽CYST和姜黄素(curcumin,Cur)为代表的亲/疏水活性物质的共载,分别提升二者生物利用度至94.83%和75.93%。同时,CYST和Cur等小分子的多羟基和多结合位点特性进一步强化了β-CD和CHC在油-水界面形成的三维网状界面膜,从而有效提升乳液稳定性。本研究可为构建功能型多糖基HIPPEs提供参考,扩宽其在食品、化妆品、涂料等领域的应用。

安宫牛黄丸治疗基底节区脑出血患者颅内高压疗效观察及对血清B型钠尿肽水平的影响

目的:观察安宫牛黄丸治疗基底节区脑出血患者颅内高压疗效及对氧化应激指标以及血清B型钠尿肽(BNP)水平的影响。方法:选取80例痰热内闭证基底结节区脑出血患者,按随机数字表法分为观察组及对照组各40例,对照组接受脑出血颅内高压常规方案治疗,观察组在对照组基础上联合安宫牛黄丸治疗。比较2组治疗前后颅内压(ICP)、平均动脉压(MAP)、心率(HR)、中医证候积分、总抗氧化能力(T-Aoc)、超氧化物歧化酶(SOD)以及BNP水平的变化。结果:治疗后,2组ICP、MAP水平均较治疗前下降(P<0.05),HR水平均较治疗前上升(P<0.05);观察组ICP、MAP水平均低于对照组(P<0.05),HR水平高于对照组(P<0.05)。治疗后,剧烈头痛、动作迟缓、嗜睡中医证候积分均较治疗前下降(P<0.05),观察组上述3项中医证候积分均低于对照组(P<0.05)。治疗后,2组T-Aoc、SOD水平均较治疗前上升(P<0.05),BNP水平均较治疗前下降(P<0.05);观察组T-Aoc、SOD水平均高于对照组(P<0.05),BNP水平低于对照组(P<0.05)。结论:安宫牛黄丸治疗痰热内闭证基底节区脑出血患者,能有效改善颅内高压,缓解氧化应激反应,促进心脏功能的恢复。

Brain-derived neurotrophic factor prevents beta-amyloid-induced apoptosis of pheochromocytoma cells by regulating Bax/Bcl-2 expression

Brain-derived neurotrophic factor was utilized in the present study to treat cell injury models induced by aggregated β-amyloid（25 35）. Methylthiazolyldiphenyl-tetrazolium bromide assay and western blot analysis showed that brain-derived neurotrophic factor provided neuroprotection against cellular apoptosis by suppressing the decline in β-amyloid（25 35）-induced cell activity and the increasing ratio of Bax/Bcl-2. After treating pheochromocytoma cells with tyrosine kinase receptor B receptor inhibitor K252a, brain-derived neurotrophic factor reverses the above- mentioned changes. The experimental findings suggested that brain-derived neurotrophic factor prevented β-amyloid peptide-induced cellular apoptosis by modulating Bax/Bcl-2 expression, and this effect was associated with binding to the specific tyrosine kinase receptor B receptor.

<i>Ratanasampil</i>(Tibetan Medicine, RNSP) Reduces <i>β</i>-Amyloid Protein (Aβ) and Pro-Inflammatory Factor Levels and Improves Cognitive Functions in Mild-to-Moderate Alzheimer’s Disease (AD) Patients Living at High Altitude

Ratanasampil (RNSP) is a traditional Tibetan medicine used for the treatment of stroke and cerebrovascular diseases. Previous discoveries that RNSP can reduce β-amyloid protein levels and increase learning and memory in Alzheimer’s mouse models (Tg2576) led us to investigate whether RNSP can improve cognitive functions in Alzheimer’s patients. In this study, 146 AD patients living in Qinghai province received either one gram or 0.33 gram daily of RNSP for 16 weeks. Placebo patients received Piracetam. Serum Aβ40 and Aβ42 levels were measured at the beginning of the study and after 4 and 16 weeks of treatment. Compared to the same group before treatment, MMSE scores, ADAS-cog scores and ADL scores were significantly improved (p 0.05, p > 0.05). After 16-week treatment, serum TNF-α, IL-1β, IL-6 and Aβ42 levels were significantly decreased (p < 0. 01) in the high-dose RNSP group, whereas no significant differences were found in the low-dose and placebo groups. The Aβ42/Aβ40 ratio was significantly decreased after 4-week and 16-week treatment in the high-dose RNSP group (p < 0. 05, p < 0.01). Furthermore, serum Aβ42 concentrations had a strong positive correlation with TNF-α, IL-1β and IL-6 levels. There were no observable adverse effects in either treatment or control groups. We conclude that further clinical trials of RNSP in Alzheimer disease are warranted.

黄葵敛肠汤保留灌肠治疗溃疡性结肠湿热内蕴证临床研究

目的探讨黄葵敛肠汤保留灌肠治疗溃疡性结肠炎(UC)湿热内蕴证的临床疗效,以及对患者脑肠肽及免疫功能指标的影响。方法选取医院2020年7月至2021年6月收治的UC湿热内蕴证患者120例,随机分为观察组和对照组,各60例。对照组患者给予美沙拉嗪肠溶片口服治疗,观察组患者给予黄葵敛肠汤保留灌肠治疗,两组患者均治疗12周。结果观察组总有效率为83.33%,显著高于对照组的73.33%(P<0.05)。治疗后,观察组主要症状腹泻、腹痛、黏液血便、里急后重评分均显著低于对照组(P<0.05);Baron内镜评分及梅奥(Mayo)评分均显著低于对照组(P<0.05);脑肠肽指标血清5-羟色胺(5-HT)和血浆P物质(SP)含量均显著低于对照组,血清生长抑制(SS)水平和血浆血管活性肠肽(VIP)含量均显著高于对照组(P<0.05);血清免疫球蛋白(Ig)A、IgG水平均显著低于对照组(P<0.05)。观察组患者不良反应发生率为6.67%,显著低于对照组的15.00%(P<0.05)。结论黄葵敛肠汤保留灌肠治疗UC湿热内蕴证疗效佳,可抑制患者的肠黏膜炎性反应,减轻黏膜损伤,且安全性好。

Determination of β-amyloid peptides in vitro aggregation process by electrochemistry

An impedimetric sensor(EA/mAb/CeZnO/GCE)for detecting β-amyloid peptides(Aβ)was successfully constructed by synthesizing ceria doped zinc oxide(CeZnO)nanoparticles to modify the glassy carbon electrode(GCE)for immobilizing the antibody mAb,and blocking the unbound carboxyl sites with ethanolamine(EA).The sensor exhibited a linear detection range of 10–100 pM and a detection limit of 1.7 pM(S/N=3),and the relative standard deviations(RSDs)of the inter-electrode and intra-electrodes were less than 3.0%.After it was stored at 4℃ for 2 d,its impedance value was 101.6% of original impedance response.The sensor was used to measure the remaining content of Aβ incubated in vitro.The results showed that after incubation for 48 h and 60 h,the content of aggregated Aβ(oligomers and fibers)almost unchanged,reaching about 90%.This method had the advantages of time-saving,simple procedure,economical,excellent conductivity,good reproducibility and stability.

穿膜肽HIV Tat蛋白的研究进展

经数十年的研究发现HIV—ITat蛋白的转导域具有穿膜活性,能将外源性的生物学分子如多肽、寡核苷酸、DNA、蛋白、质粒甚至颗粒性物质携带入多种哺乳动物活细胞质膜及核膜,并且对细胞不会产生毒副作用。尽管其穿膜途径的具体机制,目前尚不十分清楚,但却不影响其在生命科学研究领域,包括基础研究和临床研究中的应用。HIV-ITat蛋白转导域的发现及应用,为生物学研究领域开辟了一条新的探索途径。

穿膜肽的内化机制及其应用

穿膜肽是一类具有特殊穿膜功能的多肽分子,能携带其它分子甚至超分子颗粒穿膜进入细胞内部.早期研究认为,其进胞是一种无需受体、也不存在饱和状态的非经典胞吞行为.近年研究表明,其穿膜机制可能与其含有的氨基酸种类有很大关系.现在,穿膜肽的穿膜过程称为巨型胞饮行为,它与传统的胞吞形式很相似.当然,还可能存在着其它的进胞方式而没有被证明或发现.关于穿膜肽的应用也是人们最感兴趣的,在很多领域的研究都在进行并不断取得进展.不论是生物界还是医学界,穿膜肽都被认为将是一类非常有发展潜力的多肽分子.

一种用于穿透多肽筛选的随机文库的构建及筛选

以增强型绿色荧光蛋白(enhancedgreenfluorescenceprotein,EGFP)为示踪物,在pET-14b载体上构建编码12个氨基酸的随机多肽表达文库.建立一种简便、经济、有效的文库筛选方法,从所构建的文库中筛选出细胞穿透多肽(cell-penetratingpeptide,CPP).采用点突变技术,首先在pET-14b载体多克隆位点NdeⅠ和XhoⅠ之间加入4个限制性内切酶位点,随后在BamHⅠ位点后加入三联终止密码子,接着再利用亚克隆的方法在KpnⅠ和XhoⅠ之间插入EGFP,形成一个新的用于原核表达示踪蛋白的载体pET-14bMCStop/EGFP.最后再利用点突变技术在上述构建的示踪载体的多克隆位点XhoⅠ和BamHⅠ之间插入36个随机碱基序列.以His-Tat-EGFP作为工具建立有效的筛选方法,利用这种方法对文库进行筛选.酶切和测序表明,示踪载体的构建是正确的,且在大肠杆菌中可有效地表达出His标记的EGFP.在示踪载体的基础上构建的随机多肽文库至少包含了105个独立克隆,其中90%以上的克隆插入的随机片段都是36个碱基.建立的筛选方法是可行的,并用此方法进行了初步的筛选.

联合用药对阵发性心房颤动患者疗效观察

目的 观察联合应用小剂量胺碘酮、螺内酯与厄贝沙坦对非瓣膜病阵发性心房颤动患者窦性心律的维持,对左心房内径、脑钠肽以及安全性的影响.方法 将142例非瓣膜病阵发性心房颤动按就诊顺序随机分为胺碘酮组（n=47）,胺碘酮加螺内酯组（n=47）,胺碘酮、厄贝沙坦、螺内酯组（联合用药组,n=48）,3组均服用胺碘酮,胺碘酮加螺内酯组在应用胺碘酮基础上加用螺内酯,联合用药组在应用胺碘酮、螺内酯基础上加用厄贝沙坦,观察3组治疗6、12、18个月后的左心房内径、脑钠肽变化以及治疗3、6、12、18、24个月后的窦性心律维持率和安全性.结果 治疗6个月后3组左心房内径、脑钠肽值差异无统计学意义,但12个月后胺碘酮加螺内酯组、联合用药组的左心房内径、脑钠肽值明显小于胺碘酮组（P＜0.05）,联合用药组左心房内径、脑钠肽值明显小于胺碘酮加螺内酯组（P＜0.05）;治疗3、6个月后胺碘酮组窦性心律维持率低于胺碘酮加螺内酯组、联合用药组,治疗6个月后胺碘酮组和联合用药组之间差异有统计学意义（P＜0.05）,治疗12个月后胺碘酮加螺内酯组、联合用药组的窦性心律维持率明显大于胺碘酮组（P＜0.05）,联合用药组窦性心律维持率明显大于胺碘酮加螺内酯组（P＜0.05）.结论 胺碘酮、螺内酯联合治疗非瓣膜病阵发性心房颤动维持实性心律的疗效优于单用胺碘酮,并能延缓左心房的扩大和脑钠肽值的升高,在胺碘酮、螺内酯基础上加用厄贝沙坦则使上述疗效进一步加强.

食品内外部因素对金属抗菌肽SIF_(4)抑菌活性影响及生物相容性分析

为阐明食品内外部因素对金属抗菌肽SIF_(4)抑菌活性的影响规律,以金黄色葡萄球菌为指标菌,考察了温度、pH、金属阳离子、蛋白酶、反复冻融和化学试剂等因素对抑菌活性的影响,并研究了SIF_(4)的生物相容性。结果表明,金属抗菌肽SIF_(4)具有较好的热稳定性和pH稳定性,121℃仍可保持(92.68±0.40)%抑菌活性,pH 2~8可保持(99.45±0.40)%以上抑菌活性;Ca^(2+)、Mg^(2+)、K^(+)和Na^(+)等金属阳离子对抑菌活性无显著影响(P>0.05);胃蛋白酶、胰蛋白酶和蛋白酶K处理后仍可保持较高抑菌活性,具有较好的酶耐受性;6次冻融处理后抑菌活性仍高达(99.00±0.81)%,表明具有较好的冻融稳定性;EDTA和Tween-80处理对抑菌活性无显著影响(P>0.05);低浓度十二烷基硫酸钠(sodium dodecyl sulfate,SDS)和尿素处理对抑菌活性无显著影响(P>0.05),高浓度处理后抑菌活性稍有下降;SIF_(4)对血红细胞具有较好的生物相容性。研究认为,SIF_(4)对食品内外部因素变化具有较好的耐受性和较好的生物相容性,可作为一种新型抗菌剂用于食品抗菌保鲜。