Staphylococcus aureusβ-hemolysin-neutralizing single-domain antibody isolated from phage display library of Indian desert camel

Objective:To isolate and characterize Staphylococcus aureus(S.aureus)β-hemolysinneutralizing dAbs from phage display library of Indian desert camel.Methods:Phage display library of 5×10 dAb clones of LPS-immunized Indian desert camel constructed in our laboratory was used for selection of S.aureus exotoxin-specific clones by panning technique.Enrichment of Ag-specific clones in successive rounds of panning was assessed by phage-ELISA and phage titration.Different dAb clones binding to S.aureus exotoxin Ags were expressed with C-terminal 6×His tag in E.coli and purified by Ni-chelate chromatography.The expression was verified by SDS-PAGE and western blotting.The purified clones were tested for inhibition of ’hot-cold’ hemolytic activity in vitro.Resistance to thermal inactivation of the dAb clones was studied by observing the effect of heat treatment from 50℃to 99℃for 30 min on the ’hot-cold’ hemolytic activity in vitro.Results:Several dAb clones binding to S.aureus exotoxins were isolated and enriched by three rounds of panning.The soluble dAb clones were approximately～16 kDa in size and reacted with 6×His tag specific murine monoclonal antibody in western blot.One of the Ni-chelate affinity purified dAb.6×His clones,inhibited S.aureusβ-hemolysin activity in vitro and resisted thermal inactivation upto 991.Conclusions:An S.aureusβ-hemolysinneutralizing dAb clone of possible therapeutic potential has been isolated.

Effect of bacteriophage lysin on lysogens

Objective:To study the effect of phage lysin on the growth of lysogens.Methods:Sputum specimens processed by modified Petroff's method were respectively treated with phagebiotics in combination with lysin and lysin alone.The specimens were incubated at 37 ℃ for 4 days.At the end of day 1,2,3 and day 4,the specimens were streaked on blood agar plates and incubated at37 ℃ for 18-24 hours.The growth of normal flora observed after day 1 was considered as lysogens.Results:Sputum specimens treated with phagebiotics-lysin showed the growth of lysogens.When specimens treated with lysin alone,lysogen formation was avoided and normal flora was controlled.Conclusions:Lysin may have no effect on the growth of lysogens.

Molecular dissection of phage lysin PlySs2: integrity of the catalytic and cell wall binding domains is essential for its broad lytic activity

The novel phage lysin PlySs2, is reported to be highly active against various bacteria, including staphylococci, streptococci and Listeria. However, the molecular mechanisms underlying its broad lytic spectrum remain to be established. In the present study, the lytic activity of the catalytic domain(CD, PlySc) and binding specificity of the cell wall binding domain(CBD, PlySb) of PlySs2 were examined. Our results showed that PlySc alone maintains very limited lytic activity. Enhanced green fluorescent protein(EGFP)-fused PlySb displayed high binding affinity to the streptococcal strains tested, including S.suis, S.dysgalactiae, and S.agalactiae, but not staphylococci, supporting its utility as a good CBD donor for streptococcal-targeted lysin engineering. EGFP-fused intact PlySs2 similarly displayed high affinity for streptococci, but not staphylococci. Notably, four truncated PlySb fragments showed no binding capacity. These findings collectively indicate that integrity of the PlySc and PlySb domains is an essential determinant of the broad lytic activity of PlySs2.

Nε-(carboxymethyl)lysine promotes lipid uptake of macrophage via cluster of differentiation 36 and receptor for advanced glycation end products

BACKGROUND Advanced glycation end products(AGEs)are diabetic metabolic toxic products that cannot be ignored.Nε-(carboxymethyl)lysine(CML),a component of AGEs,could increase macrophage lipid uptake,promote foam cell formation,and thereby accelerate atherosclerosis.The receptor for AGEs(RAGE)and cluster of differentiation 36(CD36)were the receptors of CML.However,it is still unknown whether RAGE and CD36 play key roles in CML-promoted lipid uptake.AIM Our study aimed to explore the role of RAGE and CD36 in CML-induced macrophage lipid uptake.METHODS In this study,we examined the effect of CML on lipid uptake by Raw264.7 macrophages.After adding 10 mmol/L CML,the lipid accumulation in macrophages was confirmed by oil red O staining.Expression changes of CD36 and RAGE were detected with immunoblotting and quantitative real-time polymerase chain reaction.The interaction between CML with CD36 and RAGE was verified by immunoprecipitation.We synthesized a novel N-succinimidyl-4-18Ffluorobenzoate-CML radioactive probe.Radioactive receptor-ligand binding assays were performed to test the binding affinity between CML with CD36 and RAGE.The effects of blocking CD36 or RAGE on CML-promoting lipid uptake were also detected.RESULTS The study revealed that CML significantly promoted lipid uptake by macrophages.Immunoprecipitation and radioactive receptor-ligand binding assays indicated that CML could specifically bind to both CD36 and RAGE.CML had a higher affinity for CD36 than RAGE.ARG82,ASN71,and THR70 were the potential interacting amino acids that CD36 binds to CML Anti-CD36 and anti-RAGE could block the uptake of CML by macrophages.The lipid uptake promotion effect of CML was significantly attenuated after blocking CD36 or RAGE.CONCLUSION Our results suggest that the binding of CML with CD36 and RAGE promotes macrophage lipid uptake.

Identification of the lysine and histidine transporter family in Camellia sinensis and the characterizations in nitrogen utilization

In plants,the lysine and histidine transporter(LHT)family represent a class of proteins that mediate the uptake,translocation,and utilization of amino acids.The tea plant(Camellia sinensis)is a perennial evergreen with a relatively high level of amino acids.However,systematic identification and molecular characterization of the LHT gene family has rarely been reported in tea plants.In this study,22 CsLHTs were identified from the‘Shuchazao’genome and classified into two groups.The modeled three-dimensional structure and the conserved domains presented a high similarity among the LHTs proteins.Moreover,it was predicted that a few genes were conserved through the analysis of the physiochemical characters,structures and cis-elements in promoters.The expression patterns in tea plants revealed that CsLHT7 was mainly expressed in the roots,and CsLHT4 and CsLHT11 exhibited relatively high expression in both the roots and leaves.Moreover,the expression of all three genes could be induced by organic nitrogen.Additionally,heterogeneous expression of CsLHT4,CsLHT7 and CsLHT11 in Arabidopsis thaliana decreased the aerial parts biomass compared with that in WT plants while significantly increased the rosette biomass only for CsLHT11transgenic plants versus WT plants.Overall,our results provide fundamental information about CsLHTs and potential genes in N utilization for further analysis in tea plants.

Temperate Stutzerimonas Phage Encoding Toxin-Antitoxin System Genes Represents a Novel Genus

Stutzerimonas have been extensively studied due to their remarkable metabolic and physiological diversity.However,research on its phages is currently limited.In this study,we isolated a novel double-stranded DNA(dsDNA)phage,vB_SstM-PG1,from the marine environment that infects Stutzerimonas stutzeri G1.Its dsDNA genome is 37204 bp long with a G/C content of 64.14%and encodes 54 open reading frames.The phage possesses a tail packaging structure that is different from known Stutzerimonas stutzeri phages and exhibits structural protein characteristics similar to those of temperate phages.In addition,two genes of toxin-antitoxin system,including YdaS_antitoxin and HEPN_SAV_6107,were found in the vB_SstM-PG1 genome and play important roles in regulating host growth and metabolism.With phylogenetic tree and comparative genomic analysis,it has been determined that vB_SstM-PG1 is not closely related to any phages previously identified in the GenBank database.Instead,it has a connection with enigmatic,uncultured viruses.Specifically,the vB_SstM-PG1 virus exhibits an average nucleotide identity of over 70%with six uncultivated viruses identified in the IMG/VR v4 database.This significant finding has resulted in the identification of a novel viral genus known as Metabovirus.

Wetting alternating with partial drying during grain filling increases lysine biosynthesis in inferior rice grain

Lysine content is a criterion of the nutritional quality of rice.Understanding the process of lysine biosynthesis in early-flowering superior grain(SG)and late-flowering inferior grain(IG)of rice would advance breeding and cultivation to improve nutritional quality.However,little information is available on differences in lysine anabolism between SG and IG and the underlying mechanism,and whether and how irrigation regimes affect lysine anabolism in these grains.A japonica rice cultivar was grown in the field and two irrigation regimes,continuous flooding(CF)and wetting alternating with partial drying(WAPD),were imposed from heading to the mature stage.Lysine content and activities of key enzymes of lysine biosynthesis,and levels of brassinosteroids(BRs)were lower in the IG than in the SG at the early grainfilling stage but higher at middle and late grain-filling stages.WAPD increased activities of these key enzymes,BR levels,and contents of lysine and total amino acids in IG,but not SG relative to CF.Application of 2,4-epibrassinolide to rice panicles in CF during early grain filling reproduced the effects of WAPD,but neither treatment altered the activities of enzymes responsible for lysine catabolism in either SG or IG.WAPD and elevated BR levels during grain filling increased lysine biosynthesis in IG.Improvement in lysine biosynthesis in rice should focus on IG.

Feeding Practices and Use of Lysine and Methionine in Pigrationing on Intensified Pig Breeding in the West Center and Hauts Bassins Regions of Burkina Faso

The aim of the study was to assess feeding practices and the use of lysine and methionine in pig rationing on intensified and semi-intensive pig breeding in the Koudougou and Bobo-Dioulasso areas. To this end, a cross-sectional survey was carried out on 87 breeding in these towns. A Discriminant Factorial Analysis (DFA) confirming a k-means classification of the data collected was used to retain 71 breeding divided into three breeding classes: Class A (32.4% of breeding), Class B (14.08%) and Class C (53.52%). The results show that the majority of pig breeders were men between the ages of 36 and 59. Average herd sizes were 35 ± 28;79 ± 42 and 89 ± 21 pigs for Classes A, B and C respectively. The main breeds of pig found on the breeding were crossbred, Large white, local, Landrace and Duroc. Class A (26.1%), B (30%) and C (15.8%) breeders were familiar with both lysine and methionine. Class A breeders distributed feed staggered (65.2%) and in rations (34.8%). Lysine (13%) and methionine (8.7%) were purchased at 5250 FCFA/kg. Those in class B distributed feed staggered (50%) and in the form of rations (50%), in which they incorporated lysine (30%) and methionine (30%) purchased at a cost of 2500 FCFA/kg and 3000 FCFA/kg respectively. Rationing and staggered feeding were practiced by 23.7% and 76.3% of Class C breeders respectively. Only lysine purchased at 3400 FCFA/kg was incorporated into rations by 10.5% of breeders. The high cost of lysine and methionine was incriminated by Class A (100%), B (33.3%) and C (50%) breeders. In conclusion, intensive pig breeding, the practice of rationing and the incorporation of the amino acids lysine and methionine are of ascending importance from classes C, A to B. The high cost of feedstuffs, particularly lysine and methionine, compromises their use in rations, which could have a negative impact on expected breeding performance. The screening and use of feeds rich in and/or enriched with these amino acids, through the development or adaptation of technologies, could improve the efficiency of rations and the productivity of intensive pig breeding in Burkina Faso.

Effect of exogenous free N^(ε)-(carboxymethyl)lysine on diabetes-associated cognitive dysfunction:neuroinflammation,and metabolic disorders

Diabetes-associated cognitive dysfunction has already been attracted considerable attention.Advanced glycation end products(AGEs)from daily diets are thought to be a vital contributor to the development of this diseases.However,the effect of one of the best-characterized exogenous AGEs N^(ε)-(carboxymethyl)lysine(CML)on cognitive function is not fully reported.In the present study,diabetical Goto-Kakizaki(GK)rats were treated with free CML for 8-weeks.It was found that oral consumption of exogenous CML significantly aggravated diabetes-associated cognitive dysfunction in behavioral test.In details,exogenous CML increased levels of oxidative stress,promoted the activation of glial cells in the brain,up-regulated the release of inflammatory cytokines interleukin-6,inhibited the protein expression of the brain-derived neurotrophic factor and thus led to neuroinflammation.Furthermore,exogenous CML promoted the amyloidogenesis in the brain of GK rats,and inhibited the expression of GLUT4.Additionally,several tricarboxylic acid cycle and glutamate-glutamine/γ-aminobutyric acid cycle intermediates including pyruvate,succinic acid,glutamine,glutamate were significantly changed in brain of GK rats treated with exogenous free CML.In conclusion,exogenous free CML is a potentially noxious compounds led to aggravated diabetes-associated cognitive dysfunction which could be possibly explained by its effects on neuroinflammation,energy and neurotransmitter amino acid homeostasis.

Effects of Supplemental Glutamine and Lysine on Growth Performance of Broiler Chickens

The optimum levels of Lysine and Glutamine needed for growth performance and maintenance of the chicken broilers were evaluated in a randomized 3 × 4 factorial arrangement of dietary treatments. The battery cages measured 99 × 66 × 25 cm that can be sufficient for 5 birds. Day old Chicken broilers totaling 180 were assigned to dietary treatments comprising of 3 concentrations of Lysine (0.85, 1.14, and 1.42) each in combination with 4 concentrations of Glutamine (0, 1, 2, and 3). Each dietary treatment was replicated 3 times and each replication had 5 birds. The birds were given feed and water ad libitum with a 23-hour light regimen for a period of 4 weeks. Then, the experimental birds were evaluated for body weight gain, feed consumption, and feed conversion in order to determine their optimum requirement for dietary Lysine and Glutamine. Based on the findings of this study, the highest performance was observed in birds fed the diet supplemented with 1.42 lysine and 1% glutamine, but the highest improvement in feed conversion was observed in diet contain 1.14 and 1.42 with 1% and 3% glutamine, respectively. Birds fed 1.42 lysine and 1% glutamine had the highest total body weight gain and feed consumption. The lysine requirements in the diet for Chicken are between 1.14 and 1.42 with glutamine level of 1%.

中华乌塘鳢NK-lysin基因的克隆鉴定、表达分析及抗菌功能研究

NK-lysin是一种具有广谱抗菌活性和免疫调节功能的阳离子抗菌肽。通过分子克隆方法获得中华乌塘鳢的NK-lysin基因(BsNKL)cDNA序列。BsNKL编码152个氨基酸,包含1个信号肽、1个SapB结构域和6个保守半胱氨酸残基。基因表达分析表明,BsNKL在主要器官组织中均有表达,副溶血弧菌感染能够引起BsNKL表达量在脾脏、头肾和鳃中发生显著变化。中华乌塘鳢BsNKL抗菌肽对金黄色葡萄球菌、大肠杆菌、副溶血弧菌和哈维氏弧菌等4种细菌均具有较好的抑菌活性。腹腔注射BsNKL能够为细菌感染的中华乌塘鳢提供有效的免疫保护并显著提高其存活率。综上所述,BsNKL在中华乌塘鳢抗细菌感染天然免疫应答方面发挥重要作用。

鸡抗菌肽NK-lysin的生物信息学分析

为了解鸡内源性抗菌肽NK-lysin的基本信息,试验选择鸡NK-lysin的氨基酸序列通过软件对NK-lysin的生物信息学进行了分析。结果表明:NK-lysin的等电点为5.87,分子质量为15 231.3 u,为稳定蛋白和亲水蛋白,定位于细胞质内,有1个跨膜区和1个信号肽,没有糖基化位点,但有2个磷酸化位点,二级结构由α螺旋、延伸链、β折叠和无规则卷曲组成,存在多个抗原表位。说明鸡抗菌肽NK-lysin可作用于细胞膜或某些生物分子,使菌体死亡。

Transfer of Lysine-rich Protein Gene into Rice and Production of Fertile Transgenic Plants

Lysine-rich protein gene (lys) was cloned from Psophocarpus tetragonolobus (L.) DC. A plant expression plasmid was constructed and lys gene was under the control of maize ubiquitin promoter which is the highest efficient monocotyledon promoter. The plasmid was introduced into rice embryogenic calli by microprojectile bombardment. The regenerated fertile plants were obtained by effective selection for hygromycin B resistance. Genomic PCR and Southern blotting analyses showed that the lys gene has been integrated into rice genome. Simultaneously, the results of GUS histochemical assay demonstrated that gus report gene is also expressed in leaves, stems and roots of the transgenic rice plants. Data analysis showed that lysine content in most of the 11 transgenic plants is differently improved, and in one of them increased by 16.04%.

厚壳贻贝M7lysin分子的克隆与表达

M7 lysin位于贻贝精子顶体中,是溶解卵黄膜、促进受精作用的重要蛋白质,决定了贻贝种间精卵识别的特异性。采用同源克隆法得到厚壳贻贝M7 lysin分子,并在原核生物中对该分子进行重组表达。结果表明,扩增产物为540 bp左右的片段,进一步测序发现其开放阅读框cDNA为543 bp,与贻贝、地中海贻贝、盖勒贻贝具有较高的相似性;在线翻译所得蛋白质片段中含有180个氨基酸,分子量为20 ku,等电点为8.48;通过M7 lysin氨基酸序列构建系统进化树发现,贻贝和地中海贻贝亲缘关系最近,其次为盖勒贻贝,最后是厚壳贻贝;将M7lysin分子在E.coli Rosseta(DE3)中融合表达,得到包括载体氨基酸序列在内的25 ku蛋白质,符合预期分子质量。

小球藻病毒PBCV-1特异性溶壁酶(Lysin)的溶壁活性

从PBCV 1感染小球藻NC6 4A的细胞裂解液中提取了Lysin的粗制剂 ,酶活底物范围分析表明 ,几丁质酶、壳聚糖酶和 β 1 ,3 葡萄糖苷酶是Lysin活性的主要组成部分 ,并与小球藻细胞壁的组成成分相吻合。其中几丁酯酶和壳聚糖酶 ,特别是几丁酯酶在裂解小球藻细胞壁的过程中发挥了重要的作用。Lysin粗制剂经FPLC分离纯化得到分子量分别为 5 2kD、5 6kD的两个几丁质酶 (Chil和Chi2 )和一个分子量为 3

用Phage88快速检测结核分枝杆菌

目的 建立一种特异性强、灵敏度高的致病性分枝杆菌的检测及药敏试验方法。方法 应用可表达荧光素酶的结核分枝杆菌噬菌体 Phage88,采用生物发光方法对不同细菌的发光反应和药敏试验进行检测。结果 Phage88特异地对各种分枝杆菌发光 ,对非分枝杆菌发光值很低 ,两者差异有显著性 ,不同的分枝杆菌发光值有差异 :卡介苗的发光值最高 ,结核杆菌的发光值最低 ;在含抗结核药物的培养基中 ,耐药结核杆菌的发光强度比非耐药结核杆菌强 ,其强度有明显差异。结论 用 Phage88噬菌体检测结核分枝杆菌是一种快速、敏感的检测和药敏试验方法。

不同浓度脂多糖诱导绵羊免疫器官中NK-Lysin基因表达的研究

为研究病原体侵染动物机体时抗菌肽NK-Lysin在机体反应过程中所起到的防御作用及作用机理,本研究通过实时荧光定量PCR检测NK-Lysin在脂多糖(lipopolysaccharide,LPS)不同诱导条件下及不同免疫器官中的相对表达量,探讨绵羊抗菌肽NK-Lysin在免疫器官的生成和应答反应。构建目标基因NK-Lysin和内参基因GAPDH的克隆载体,绘制出标准曲线,检测NK-Lysin的表达量。结果显示,在LPS浓度为0.01、0.1、1、10μg/mL时,NK-Lysin mRNA表达量均在8h内达到最大值,NK-Lysin基因的表达量与LPS的浓度不存在正相关性;NK-Lysin mRNA在肩前淋巴结、脾脏及血液中均有不同程度的表达,脾脏中表达量与其他组织中的表达量相比差异极显著(P<0.01)。免疫器官能在较短时间内表达出抗菌肽NKLysin,使其参与机体先天性免疫应答反应。

Poly-L-Lysine玻片在寡核苷酸芯片制备中的改进

目的为了制得适合固定未修饰寡核苷酸的芯片,提高检测灵敏性,对Patrick Brown 实验室的多聚左旋赖氨酸包被玻片的方法进行改进。方法玻片经清洗后用缩水甘油-丙氧基三甲氧基硅烷进行硅烷化,然后应用Poly-L-Lysine在玻片表面形成聚合物涂层,经次亚苯基二异硫氰酸盐表面活化后可使寡核苷酸共价连接在芯片表面。设计了各种实验考察方法改进前后芯片表面的性能,并将改进后的玻片初步应用于SARS冠状病毒寡核苷酸芯片检测中。结果方法改进后芯片表面性能优良:固定效率高、点的同一性好、杂交效率和热稳定性好、寡核苷酸结合牢固、芯片可以重复利用。结论利用共价连接,方法改进后的芯片表面适合固定未修饰的寡核苷酸,解决了寡核苷酸与玻片之间物理结合不稳定、易剥离的缺陷,提高了芯片检测的灵敏性。

Rapid Determination of Lysine Content in Maize Kernel by Amino-acid Analyzer

In this study, nine high-lysine maize kernels and two kernels of common maize hybrid were used as experiment materials, and quantitative determination of lysine contents in high-lysine maize kernels and common maize kernels was carried out using Hitachi L-8900 Automatic Amino-acid Analyzer, to know the effect of the Analyzer in distinguishing the lysine contents between common maize kernels and high-lysine maize kernels. The results showed that the lysine contents of high-lysine maize kernels were among 0.34%-0.42%, while of common maize kernels were a- mong 0.24%-0.25%, and the difference was significant. Compared with other tradi- tional methods, this method is rapid, simple, sensitive, highly reproductive and needs fewer maize kernels, thus it is applicable in maize breeding.

噬菌体多肽phage20抗胃癌肝转移作用的实验研究

目的 鉴定选择性噬菌体多肽phage2 0是否具有抗胃癌肝高转移细胞XGC9811-L肝转移的作用。方法 采用胃浆膜下裸鼠原位接种转移模型 ,观察phage2 0对胃癌细胞XGC9811-L肝转移能力的影响。裸鼠随机分为实验组 (phage2 0孵育组 12只 ) ,对照组 (XGC9811-L组 12只、M13wt孵育组 12只 ) ,原位接种后 10天、5周解剖裸鼠 ,观察肝转移率、转移瘤的数目及原发瘤的体积。结果 在原位接种后 10天 ,各组未发现肝转移灶 ,接种后5周phage2 0孵育组、XGC9811-L未孵育组、M13孵育组肝转移率分别为 :2 0 % ,10 0 % ,80 % ;肝转移灶的数目为 0 ,9.5± 2 .8,7.1± 4 .71;原发瘤的体积为 0 .6 5± 0 .4 32 ,0 .5 2 8± 0 .2 96 ,0 .5 82± 0 .348。与对照组相比phage2 0孵育组的肝转移率和转移灶的数目明显减少 ,P <0 .0 5 ,但在原发瘤的体积方面 3者间无显著差别 P >0 .0 5。结论 phage2 0可抑制胃癌肝高转移潜能细胞XGC9811-L肝转移能力 ,但对其生长无影响。