硝酸稀土促进布鲁氏菌生长试验

我们用硝酸稀土培养基做了猪种、羊种布鲁氏菌促进生长试验,结果表明,猪种布鲁氏菌菌体细胞每分裂一次其时间可提前22分钟,羊种布鲁氏菌则可提前12.7～17.5分钟,说明硝酸稀土有促进布鲁氏菌生长作用。     

快速免疫诊断牛布鲁氏菌病新方法的研究

目的建立一种牛布鲁氏菌病快速免疫诊断方法。方法斑点金免疫渗滤法(DIGFA)以硝酸纤维素膜为载体,胶体金标记蛋白为显示剂,抗原、抗体及胶体金标记蛋白通过渗滤发生反应,阳性结果在膜上显示红色斑点。结果DIGFA快速检测牛布鲁氏菌病具有很好的重现性和稳定性;敏感性、特异性分别达到97%和100%。结论DIGFA快速检测牛布鲁氏菌病,方法快速简便,测试过程1~2 min内完成,试剂敏感、特异、稳定,非常适合于布鲁氏菌病的诊断及流行病学调查。     

布鲁氏菌S2菌壳的安全性和免疫学特性研究

目的利用小鼠模型对布鲁氏菌菌壳、布鲁氏菌S2活菌和福尔马林灭活菌的安全性和免疫学特性进行比较研究。方法利用猪布鲁氏菌S2株和重组裂解质粒制备出布鲁氏菌菌壳,将布鲁氏菌菌壳、布鲁氏菌S2活菌和福尔马林灭活菌通过腹腔注射免疫小鼠,进行安全性比较分析。监测免疫后小鼠的血清抗体水平、脾脏T淋巴细胞分型,并进行免疫攻毒实验。结果与布鲁氏菌弱毒疫苗菌株S2比较,布鲁氏菌菌壳具有更好的安全性,免疫小鼠后能产生与弱毒菌株相似的血清抗体水平、脾CD3+和CD4+T淋巴细胞反应,并具有与之相当的免疫保护作用。结论布鲁氏菌菌壳具有良好的安全性,能刺激机体产生体液免疫和细胞免疫反应,可作为预防布鲁氏菌病的新型候选疫苗。     

布鲁氏菌外膜蛋白 VirB4在感染胚胎滋养层细胞中的作用分析

目的 筛选布鲁氏菌侵染牛胚胎滋养层细胞过程中与Ⅳ型分泌系统VirB4蛋白结合的潜在靶蛋白。方法 设计引物并PCR扩增布鲁氏菌的virB4基因,构建表达载体pGBKT7-VirB4,酶切鉴定,测序分析正确后,转化酿酒酵母菌感受态细胞Y187,进行自激活和毒性检测;建立布鲁氏菌侵染牛胚胎滋养层细胞模型,构建布鲁氏菌侵染牛胚胎滋养层细胞cDNA文库;采用酵母双杂交技术筛选与VirB4相互作用的滋养层细胞蛋白,实时定量PCR检测靶蛋白表达量的变化。结果 成功构建了pGBKT7-virB4诱饵质粒,转入Y187后无毒性,不能自激活;获得了布鲁氏菌侵染牛胚胎滋养层细胞cDNA文库;筛选到了13个阳性质粒,其中蛋白辅酶Q₁₀和SLC3A2在布鲁氏菌侵染后mRNA表达量均增加。结论 本试验对VirB4蛋白与宿主细胞的互作研究为进一步阐明布鲁氏菌感染宿主细胞的发病机制奠定了基础。     

噬菌体展示布鲁氏菌蛋白文库构建及差减筛选融合蛋白

目的 建立噬菌体展示羊型布鲁氏菌16M株表面蛋白文库,差减筛选具有良好特异性、亲和力及反应原性的蛋白,为确定新型诊断用抗原奠定基础。方法 利用噬菌粒载体pYW01构建重组羊型布鲁氏菌16M株基因文库,辅助噬菌体VCSM13感染基因文库进而构建噬菌体展示羊型布鲁氏菌16M蛋白文库。利用PEG纯化后,扩增蛋白文库。随机挑取单克隆,提取质粒,测序鉴定蛋白文库。通过差减筛选,选择具有良好特异性、亲和力及反应原性的表面蛋白,为确定诊断用抗原奠定基础。结果 通过DNA重组技术,构建羊型布鲁氏菌16M基因文库,库容量达到10⁸ pfu/ mL,并且随机性良好。利用辅助噬菌体VCSM13包装基因文库,随机挑取蛋白文库中单克隆,提取质粒,经测序鉴定,成功构建噬菌体展示羊型布鲁氏菌16M株表面蛋白文库。利用免疫血清及感染血清对噬菌体展示蛋白文库进行差减淘选,筛选出6个噬菌体融合蛋白。经Western-blot分析6个噬菌体融合蛋白均具有较好的反应原性及特异性。结论 成功构建噬菌体展示布鲁氏菌蛋白文库,差减筛选出6个具有较好反应原性及特异性的融合蛋白,为后续新型血清学诊断制剂筛选奠定基础。     

布鲁氏菌自杀载体的改建及其在突变株构建中的应用

目的:改建布鲁氏菌自杀载体.方法:用NdeⅠ将pKOBEG-SacB质粒中的反向筛选基因SacB切下来,与同样酶切处理的pUC19质粒连接,得到pUC19-SacB,经测序和蔗糖筛选实验证实.结果:成功将SacB从pKOBEG-SacB中切下来并插入到pUC19质粒中,构建得到了pUC19- SacB.测序和蔗糖筛选实验证实.插入的sacB基因具有较好的反筛功能.我们利用该质粒,成功的构建了布鲁氏菌Ⅳ型分泌系统的突变株,改建的载体能高效的应用于布鲁氏菌染色体的精确修饰.结论:构建了一个新的自杀质粒pUC19-SacB,该质粒能用于布鲁氏菌无痕缺失突变株的构建.     

布鲁氏菌非编码小RNA BSR1526突变株与过表达株的构建及毒力研究

目的 为探讨非编码小RNA BSR1526在布鲁氏菌胞内生存中的作用,构建BSR1526突变株与过表达株并分析它们在模拟胞内环境和小鼠体内的存活能力。方法 首先采用Northern blot和RT-PCR验证布鲁氏菌16M在体外刺激条件中BSR1526的转录;然后采用融合PCR构建BSR1526缺失株;将BSR1526插入到质粒pBBR1-MCS4中,电转入16M中构建过表达株16M-BSR1526;最后分析BSR1526缺失株和过表达株在模拟胞内环境以及小鼠体内的存活力。结果 BSR1526在布鲁氏菌16M中存在转录,且在不同刺激条件下转录水平不同。BSR1526缺失或过表达影响了布鲁氏菌16M在体外的生长能力,缺失后在热应激、氧化压力及酸性环境下的生存能力下降,在小鼠脾脏内的细菌数量显著下降,表明BSR1526影响了布鲁氏菌的毒力。结论 非编码小RNA BSR1526影响着布鲁氏菌16M在胞内生存能力,缺失BSR1526后布鲁氏菌16M抵抗外界不良环境的能力下降,同时在小鼠体内的毒力下降。     

量子点标记免疫层析技术检测布鲁氏菌病的方法研究

目的 通过研究量子点标记免疫层析技术检测布鲁氏菌病抗体,制备量子点免疫层析试纸条,验证快速检测布鲁氏菌病的可行性。方法 将蛋白A和布鲁氏菌全菌蛋白分别作为质控线和检测线喷涂在硝酸纤维素膜上,以量子点标记布鲁氏菌全菌蛋白,稀释后喷涂在玻璃纤维素膜上,最后进行组装、切割制成检测用试纸条。应用该试纸条检测布鲁氏菌病患者样本102例,健康人血清47例,以试管凝集试验为对照,比较两组方法的符合率。结果 建立的量子点免疫层析试纸条性能较好,布鲁氏菌病抗体的检测敏感性为99.0%,特异度为95.8%,两者符合率为98.0%;将阳性血清稀释至128倍,通过荧光检测仪仍可检测到T线荧光值,该纸条重复性好,储存时间30 d内稳定性较好,其变异系数为4.1%。结论 该方法操作简单、快速稳定、灵敏度高、成本低,15 min内完成检测,血清用量低,可实现现场快速检测,在布鲁氏菌病的早期检测中具有良好前景。     

人工诱变利福平抗性布鲁氏菌转录组测序分析

目的 筛选布鲁氏菌中参与利福平代谢相关基因(除rpoB基因外)。方法 利用人工诱变技术获得利福平抗性菌株,通过转录组测序获得标准菌株和利福平抗性菌株全基因组水平的基因表达量,利用EBSeq算法筛选差异表达基因,预测利福平代谢相关基因及主要代谢途径。结果 通过不同浓度的利福平人工诱变,能够获得抗性表型稳定的布鲁氏菌变异菌株。转录组测序发现利福平抗性菌株中有121个基因表达量上调,197个基因表达量下调,差异基因功能主要集中于催化活性、细胞膜和细胞成分、代谢过程和细胞过程;主要参与抗生素的生物合成、细菌分泌系统和ABC 转运蛋白等代谢通路。结论 包括virB操纵子在内的涉及碳代谢等代谢通路的基因,通过表达量的改变参与抵抗利福平的作用。 本研究为布鲁氏菌耐药相关基因的筛选提供新思路,同时为布鲁氏菌耐药菌株的防控提供基础性数据。     

布鲁氏菌病脊柱炎药物治疗的影响因素分析

目的 通过对布鲁氏菌病脊柱炎患者临床特征和转归的分析,明确布鲁氏菌病脊柱炎转归的影响因素.方法 选取2016年5月-2018年5月就诊于我院的81例布鲁氏菌病脊柱炎患者为研究对象.回顾性分析患者病程,基础疾病及神经功能损伤等情况及实验室和影像学检查结果,通过单因素分析和多因素Logistic回归分析,筛选出影响药物治疗...     

Cloning and nitrate induction of nitrate reductase mRNA

Nitrate is the major source of nitrogen taken from the soil by higher plants but requires reduction to ammonia prior to incorporation into amino acids. The first enzyme in the reducing pathway is a nitrate-inducible enzyme, nitrate reductase (EC 1.6.6.1). A specific polyclonal antiserum raised against purified barley nitrate reductase has been used to immunoprecipitate in vivo labeled protein and in vitro translation products, demonstrating that nitrate induction increases nitrate reductase protein and translatable mRNA. A partial cDNA clone for barley nitrate reductase has been isolated and identified by hybrid-selected translation. RNA blot-hybridization analysis shows that nitrate induction also causes a marked increase in the steady-state level of nitrate reductase mRNA.     

Genetic identification of a gene involved in constitutive, high-affinity nitrate transport in higher plants.

Two mutations have been found in a gene (NRT2) of Arabidopsis thaliana that specifically impair constitutive, high-affinity nitrate uptake. These mutants were selected for resistance to 0.1 mM chlorate in the absence of nitrate. Progency from one of the backcrossed mutants showed no constitutive uptake of nitrate below 0.5 mM at pH 7.0 in liquid culture (that is, within 30 min of initial exposure to nitrate). All other uptake activities measured (high-affinity phosphate and sulfate uptake, inducible high-affinity nitrate uptake, and constitutive low-affinity nitrate uptake) were present or nearly normal in the backcrossed mutant. Electrophysiological analysis of individual root cells showed that the nrt2 mutant showed little response to 0.25 mM of nitrate, whereas NRT2 wild-type cells showed an initial depolarization followed by recovery. At 10 mM of nitrate both the mutant and wild-type cells displayed similar, strong electrical responses. These results indicate that NRT2 is a critical and perhaps necessary gene for constitutive, high-affinity nitrate uptake in Arabidopsis, but not for inducible, high-affinity nor constitutive, low-affinity nitrate uptake. Thus, these systems are genetically distinct.     

Sucrose mimics the light induction of Arabidopsis nitrate reductase gene transcription.

Nitrate reductase, the first enzyme in nitrate assimilation, is located at the crossroad of two energy-consuming pathways: nitrate assimilation and carbon fixation. Light, which regulates the expression of many higher-plant carbon fixation genes, also regulates nitrate reductase gene expression. Located in the cytosol, nitrate reductase obtains its reductant not from photosynthesis but from carbohydrate catabolism. This relationship prompted us to investigate the indirect role that light might play, via photosynthesis, in the regulation of nitrate reductase gene expression. We show that sucrose can replace light in eliciting an increase of nitrate reductase mRNA accumulation in dark-adapted green Arabidopsis plants. We show further that sucrose alone is sufficient for the full expression of nitrate reductase genes in etiolated Arabidopsis plants. Finally, using a reporter gene, we show that a 2.7-kilobase region of 5' flanking sequence of the nitrate reductase gene is sufficient to confer the light or the sucrose response.     

Rationale for intermittent nitrate therapy.

Tolerance to the pharmacologic and therapeutic effects of nitrate therapy is now well established. This phenomenon may be defined as either a decreased response to a given amount of nitrate or the need for an increased amount of nitrate to maintain a constant effect. Tolerance has been demonstrated with all forms of nitrate therapy that maintain continuous blood levels of the drug, including frequent oral dosing, constant intravenous infusion, and continuous transdermal delivery. It can develop rapidly after only a few doses of a nitrate preparation and tends to be partial rather than absolute. Strategies for the prevention of nitrate tolerance include the avoidance of maximum nitrate doses and the use of intermittent nitrate dosing regimens. Providing a relatively brief nitrate-free interval restores vascular responsiveness to nitrates, most likely due to a recovery of the metabolic mechanisms responsible for the therapeutic effect of these drugs. The duration of this period of nitrate abstinence varies, depending on the nitrate preparation used but is generally in the range of 8-12 hours. Such intermittent therapy not only reduces the risk of nitrate tolerance, but also provides a convenient approach to outpatient management.     

Roles of dietary inorganic nitrate in cardiovascular health and disease

Inorganic nitrate from dietary and endogenous sources is emerging as a substrate for in vivo generation of nitric oxide (NO) and other reactive nitrogen oxides. Dietary amounts of nitrate clearly have robust NO-like effects in humans, including blood pressure reduction, inhibition of platelet aggregation, and vasoprotective activity. In animal models, nitrate protects against ischaemia-reperfusion injuries and several other types of cardiovascular disorders. In addition, nitrate most surprisingly decreases whole body oxygen cost during exercise with preserved or even enhanced maximal performance. Oxidative stress and reduced NO bioavailability are critically linked to development of hypertension and other forms of cardiovascular diseases. Mechanistically, a central target for the effects of nitrate and its reaction products seems to be the mitochondrion and modulation of oxidative stress. All in vivo effects of nitrate are achievable with amounts corresponding to a rich intake of vegetables, which are particularly rich in this anion. A theory is now emerging suggesting nitrate as an active component in vegetables contributing to the beneficial health effects of this food group, including protection against cardiovascular disease and type-2 diabetes.     

Nitrate reduction as a marker for hypoxic shiftdown of Mycobacterium tuberculosis.

SETTING: In vitro cultures. OBJECTIVE: To characterize nitrate reduction during aerobic growth and hypoxic shiftdown to non-replicating persistence of Mycobacterium tuberculosis cultures. DESIGN: The rates of reduction of nitrate to nitrite were measured in cultures of M. tuberculosis growing aerobically or undergoing hypoxic shiftdown. RESULTS: Tubercle bacilli growing aerobically in the presence of nitrate reduce nitrate at a rate proportional to the substrate concentration, continuing until the substrate is exhausted. When the bacilli in an oxygen restricted model enter microaerophilic non-replicating persistence (NRP) stage 1, they exhibit a marked increase in rate of nitrate reduction that is independent of substrate concentration, and terminates by feedback inhibition when the concentration of nitrite produced approaches 2.5 mM. When bacilli in the oxygen restricted model are not supplemented with nitrate until they enter microaerophilic NRP stage 1, they exhibit an induction period before the rapid nitrate reduction starts. When the nitrate is not added until the bacilli have entered the anaerobic NRP stage 2, reduction of the substrate starts immediately. Nitrite is not reduced by M. tuberculosis in any stage of its growth or NRP. CONCLUSION: The hypoxically induced nitrate reduction probably serves a respiratory function in supporting hypoxic shiftdown of M. tuberculosis from aerobic growth to non-replication persistence and represents a useful new marker for monitoring that shiftdown. This response may help the bacilli survive in oxygen depleted regions of inflammatory or necrotic tissue, where nitrate can occur as a degradation product of nitric oxide.     

Nitrate reduction as a marker for hypoxic shiftdown of Mycobacterium tuberculosis

Setting : In vitro cultures.Objective : To characterize nitrate reduction during aerobic growth and hypoxic shiftdown to non-replicating persistence of Mycobacterium tuberculosis cultures.Design : The rates of reduction of nitrate to nitrite were measured in cultures of M. tuberculosis growing aerobically or undergoing hypoxic shiftdown.Results : Tubercle bacilli growing aerobically in the presence of nitrate reduce nitrate at a rate proportional to the substrate concentration, continuing until the substrate is exhausted. When the bacilli in an oxygen restricted model enter microaerophilic non-replicating persistence (NRP) stage 1, they exhibit a marked increase in rate of nitrate reduction that is independent of substrate concentration, and terminates by feedback inhibition when the concentration of nitrite produced approaches 2.5 mM. When bacilli in the oxygen restricted model are not supplemented with nitrate until they enter microaerophilic NRP stage 1, they exhibit an induction period before the rapid nitrate reduction starts. When the nitrate is not added until the bacilli have entered the anaerobic NRP stage 2, reduction of the substrate starts immediately. Nitrite is not reduced by M. tuberculosis in any stage of its growth or NRP.Conclusion : The hypoxically induced nitrate reduction probably serves a respiratory function in supporting hypoxic shiftdown of M. tuberculosis from aerobic growth to non-replication persistence and represents a useful new marker for monitoring that shiftdown. This response may help the bacilli survive in oxygen depleted regions of inflammatory or necrotic tissue, where nitrate can occur as a degradation product of nitric oxide.     

Therapeutic effects of inorganic nitrate and nitrite in cardiovascular and metabolic diseases

Nitric oxide (NO) is generated endogenously by NO synthases to regulate a number of physiological processes including cardiovascular and metabolic functions. A decrease in the production and bioavailability of NO is a hallmark of many major chronic diseases including hypertension, ischaemia–reperfusion injury, atherosclerosis and diabetes. This NO deficiency is mainly caused by dysfunctional NO synthases and increased scavenging of NO by the formation of reactive oxygen species. Inorganic nitrate and nitrite are emerging as substrates for in vivo NO synthase‐independent formation of NO bioactivity. These anions are oxidation products of endogenous NO generation and are also present in the diet, with green leafy vegetables having a high nitrate content. The effects of nitrate and nitrite are diverse and include vasodilatation, improved endothelial function, enhanced mitochondrial efficiency and reduced generation of reactive oxygen species. Administration of nitrate or nitrite in animal models of cardiovascular disease shows promising results, and clinical trials are currently ongoing to investigate the therapeutic potential of nitrate and nitrite in hypertension, pulmonary hypertension, peripheral artery disease and myocardial infarction. In addition, the nutritional aspects of the nitrate–nitrite–NO pathway are interesting as diets suggested to protect against cardiovascular disease, such as the Mediterranean diet, are especially high in nitrate. Here, we discuss the potential therapeutic opportunities for nitrate and nitrite in prevention and treatment of cardiovascular and metabolic diseases.     

Validation of nitrite and nitrate measurements in exhaled breath condensate

BACKGROUND: Inflammatory markers in exhaled breath condensate (EBC) are investigated as a non-invasive approach to monitoring of inflammation in the respiratory tract. EBC concentrations of nitrite and nitrate, the stable end products of oxidative metabolism of nitric oxide, are increased in patients with asthma, especially during acute exacerbations. OBJECTIVES: To examine methodological aspects of nitrite and nitrate measurements in EBC such as sample collection, storage and analysis. METHODS: In a randomized study, EBC was collected twice within 1 h (with and without a nose clip) in 20 healthy adults and 20 patients with well-controlled asthma and no symptoms of allergic rhinitis. Nitrite and nitrate were assayed by ionex chromatography and fluorimetrically after derivatization with diaminonaphthalene. RESULTS: The geometric mean [exp (mean +/- SD)] EBC levels of nitrite and nitrate in healthy subjects [4.3 (3.0-6.1) and 11.0 (5.3-22.7) micromol/l] and patients [4.6 (2.6-7.3) and 8.7 (3.2-23.8) micromol/l] did not differ (p = 0.13). Wearing a nose clip (p = 0.3) did not influence nitrite and nitrate concentrations. The mean intra-subject %CVs of EBC concentrations of nitrite were 26 and 21% in healthy subjects and patients, while those of nitrate achieved 49 and 88%, respectively. CONCLUSIONS: Ionex chromatography of nitrite and nitrate requires no sample pretreatment and provides comparable results as a more laborious diaminonaphthalene method. EBC samples should be kept cold (8 degrees C) and analyzed for nitrite and nitrate within 24 h of collection or stored in the freezer and thawed preferably only once. Wearing a nose clip during EBC collection has no influence on nitrite and nitrate concentrations. Short-term repeatability of nitrite and nitrate measurements was worse compared to published data on exhaled nitric oxide.