猪链球菌1、2、14、1/2型荚膜多糖的单糖组成比较

【目的】猪链球菌1、2、14和1/2型间存在单向或双向的交叉抗原性,这种交叉抗原性的产生原因至今未被揭示。【方法】采用Sephacryl S-300凝胶层析柱对猪链球菌14和1/2型荚膜多糖进行分离纯化,经苯酚-硫酸检测和dot-ELISA辅助鉴定,确定荚膜多糖成分。采用高效凝胶渗透色谱法测定14和1/2型猪链球菌荚膜多糖分子量分别为487.38 kDa和512.72 kDa。【结果】经柱前衍生高效液相色谱法、荧光标记液相色谱法和核磁共振测定14和1/2型猪链球菌荚膜多糖单糖组成分别为:Glc/Gal/GlcNAc/Rha/Neu5Ac(1∶2.94∶1.35∶0.24∶0.37)和Glc/Gal/GlcNAc/GalNAc/Rha/Neu5Ac(1∶1.67∶1.05∶0.93∶0.72∶0.7)。并与已知的猪链球菌1、2型荚膜多糖的单糖组成进行比较分析,发现4种血清型荚膜多糖都具有Glc、GlcNAc、Gal和Neu5Ac,但单糖组成和比列并无明显相似性,这种交叉抗原性可能是由于荚膜多糖的空间结构相似性和(或)细胞表面的其他成分引起的。     

肺炎链球菌cps操纵子的启动子序列点突变可导致细菌荚膜缺失

摘要:【目的】明确肺炎链球菌荚膜多糖合成操纵子的启动子序列出现的点突变313713 T→C是否可导致细菌荚膜缺失。【方法】Western blot检测肺炎链球菌突变株SPY1 (NC_008533.1 313713 T→C)荚膜多糖含量;实时定量荧光PCR分析荚膜多糖合成基因cps2A、cps2B、cps2C以及cps2D的表达量;构建重组质粒pEVP3-cps promoterD39和pEVP3-cps promoterSPY1,分别转化D39和SPY1菌株,通过β-半乳糖苷酶活性检测来验证转入的启动子序列对细菌荚膜合成的影响,并通过电镜观察荚膜结构和荚膜肿胀试验进一步验证。【结果】肺炎链球菌SPY1 的荚膜多糖含量较野生型显著下降,其相关基因cps2A、cps2B、cps2C以及cps2D的表达量较野生型D39均显著降低;与D39-pEVP3-cps promoterD39对比,D39-pEVP3-cps promoterSPY1的β-半乳糖苷酶活性下降了76%,与SPY1-pEVP3-cps promoterD39相比,SPY1-pEVP3-cps promoterSPY1的β-半乳糖苷酶活性下降了约79%;电镜结果显示,重组SPY1-pEVP3-cps promoterD39荚膜可恢复至野生型水平,并且重组D39-pEVP3-cps promoterSPY1 (NC_008533.1 313713 T→C)荚膜肿胀试验呈阴性。【结论】荚膜多糖合成操纵子的启动子序列点突变313713 T→C可导致荚膜多糖合成基因表达显著下调,从而引起菌株SPY1的荚膜显著减少甚至缺失。     

不同培养条件对胶质芽孢杆菌诱导碳酸钙晶体形成的影响

【目的】研究不同培养条件对胶质芽孢杆菌(Bacillus mucilaginosus)菌体形态、数量和分泌的碳酸酐酶(CA酶)活性的影响,以及不同方式培养的菌体与碳酸钙晶体的生长及其形貌、数量之间的联系。【方法】分别采用无氮和有氮培养基培养胶质芽孢杆菌,进行菌体形态、数量及CA酶活性的比较,收集不同培养方式的菌体加入碳酸钙结晶体系中以研究细菌与碳酸钙晶体形成的联系。【结果】在无氮培养条件下,胶质芽孢杆菌数量少、荚膜肥厚,细菌培养液CA酶活力较低;有氮培养条件下,菌体数量多、荚膜单薄,细菌培养液CA酶活力较高。在碳酸钙结晶体系中加入无氮培养的菌体,生成的碳酸钙晶体表面光滑,体积较大但数量较小,加入有氮条件下培养的菌体形成的碳酸钙晶体表面粗糙,数量大但体积较小。【结论】不同培养条件能够引起胶质芽孢杆菌菌体数量、荚膜多糖及CA酶活的明显差异,进而对碳酸钙晶体的生成和形貌产生影响。     

褐藻胶降解菌的筛选、鉴定及产酶条件优化

【目的】筛选一株能降解褐藻胶的菌株,并优化产酶条件以提高褐藻胶裂解酶活力。【方法】从漳州海域采集到海水和海泥,以海藻酸钠为唯一碳源,通过富集培养、初筛、复筛筛选到一株能够降解褐藻胶的菌株。依据16S rRNA序列分析、生理生化特征、菌体形态及菌落特征对该菌进行鉴定。通过单因素和正交试验对该菌的产酶条件进行优化。【结果】该菌属于海科贝特氏菌,命名为Cobetiamarina HQZ08。该菌株最佳的产酶培养基组成为:海藻酸钠7.00g/L、蛋白胨3.00g/L、NaCl30.00g/L,K2HPO4·3H2O 1.25 g/L。最佳发酵条件为:接种量2%,接种龄12 h,培养基起始pH为7.0,培养温度25°C,培养时间24 h。优化后褐藻胶裂解酶活力达到68.5 U/mL,TLC法分析酶解产物为褐藻胶寡糖。【结论】HQZ08菌株可以用于降解褐藻胶,产生聚合度为2–6的褐藻胶寡糖。     

肌苷生产菌枯草芽孢杆菌ATCC 13952的全基因组测序及序列分析

摘要:【目的】枯草芽孢杆菌ATCC 13952是一株肌苷工业生产菌株。为深入研究ATCC 13952菌株积累肌苷的分子机制以及为进一步分子育种研究提供序列背景信息,有必要解析ATCC 13952菌株的基因组序列信息。【方法】本研究采用高通量测序和Sanger测序相结合对ATCC 13952菌株进行全基因组测序,然后使用相关软件对测序数据进行基因组组装、基因预测与功能注释、GO/COG 聚类分析、共线性分析等。【结果】枯草芽孢杆菌ATCC 13952整个基因组大小为3876276 bp,GC含量为45.8%,序列已提交至GenBank 数据库,登录号为CP009748。比较基因组及嘌呤代谢相关基因分析结果显示:枯草芽孢杆菌ATCC 13952与其他几株芽孢杆菌具有较好的基因组共线性关系,嘌呤代谢相关基因编码的蛋白与标准菌株比较发生了一些缺失和突变。【结论】本研究首次报道了一株肌苷生产菌枯草芽孢杆菌ATCC 13952的全基因组序列,分析了基因组基本特征,初步探讨了该菌株积累肌苷的分子机制,为后续的进一步分子育种提供了理论基础。     

植物乳杆菌荚膜缺陷型菌株的诱变选育

以自主分离和鉴定的产荚膜多糖植物乳杆菌C88为出发菌株,采用亚硝基胍诱变、墨汁负染和显微镜观察筛选获得一株荚膜缺陷型突变株,命名为植物乳杆菌C88M3,经多次传代突变菌株具有良好的遗传稳定性。通过16SrDNA序列分析、菌株生长曲线和RAPD分析比较了野生型菌株和荚膜缺陷型菌株在遗传特性和产荚膜情况方面的差异。通过化学诱变方法获得了乳杆菌荚膜缺陷型菌株,对进一步研究荚膜多糖在乳杆菌益生性中的功能和作用机制具有重要意义。     

啤酒酵母工业菌株单倍体的诱导、分离和鉴定

【目的】探索适宜的方法进行啤酒酵母工业菌株单倍体的诱导、分离和鉴定,为啤酒酵母改良和遗传学研究提供便利。【方法】首先,选择产孢效果最好的培养基进行产孢诱导,诱导产生的孢子在YPD培养基上形成菌落后,用流式细胞技术检测其DNA含量,进而判断其倍性;单倍体菌株的交配型通过MAT-PCR和杂交实验确定。【结果】啤酒酵母工业菌G-03通过产孢诱导和孢子分离、富集后得到26株菌,最终通过流式细胞技术确定了其中4株为单倍体,MATa和MATα型各2株。通过扫描电镜法观察4株单倍体菌株及出发菌G-03的细胞形态,发现单倍体菌株的形态和出发菌有较大区别,单倍体菌株长期培养没有假丝生长的现象发生。【结论】啤酒酵母工业菌单倍体育种较为困难,严格的单倍体筛选、鉴定尤其具有挑战性。     

吸水链霉菌ATCC 29253产Hygrocin A发酵条件的优化

【背景】Hygrocins是一种萘安莎抗生素,具有良好的新药开发潜能。但在常见培养基及发酵条件下菌体内Hygrocin A含量一般很低,甚至难以直接进行准确检测。【目的】提高吸水链霉菌ATCC 29253发酵物中Hygrocin A的产量。【方法】采用单因素与正交试验设计优化相结合的方法系统考察碳源、氮源、磷酸盐、MgCl_2浓度、NaCl浓度、种子菌龄等因素对吸水链霉菌ATCC 29253产Hygrocin A能力的影响。【结果】最佳发酵条件为(g/L):葡萄糖4.0,黄豆饼粉8.0,麦芽提取物10.0,K_2HPO_4 1.5,KH_2PO_4 1.5,NaCl 1.5,Mg Cl2 1.0;种子最佳活化时间为48 h;培养参数:摇床转速200 r/min,初始pH为6.8-7.0,瓶装量50 m L/250 m L,接种量5%,30°C培养10 d。在优化条件下,Hygrocin A产量与其原始培养基M10相比提高了500%,Rapamycin产量同时下降了95%。【结论】通过培养基优化,可显著提高吸水链霉菌ATCC 29253中Hygrocin A产量,为Hygrocin A合成应用研究奠定基础,同时可使Rapamycin产量明显下降。这说明可通过选择培养条件有目的地调节两种抗生素的代谢通量,进而开展多种抗生素同时表达的代谢调控研究。     

真菌Talaromyces stipitatus WH4-2中的抗菌活性成分

【目的】对真菌菌株WH4-2进行鉴定并分离鉴定具有抗菌活性的天然产物。【方法】综合菌落形态和ITS全序列分析,鉴定菌株种属。以抗菌活性分析为导向对其中的活性组分进行分离纯化,波谱分析鉴定结构。采用微量稀释法对化合物进行活性复筛,得最小抑菌浓度MIC值。【结果】真菌菌株WH4-2鉴定为Talaromyces stipitatus。从其土豆发酵液中分离鉴定化合物1 (5S-arugosin K)、2 (5R-arugosin K)、3 (5S-arugosin M)、4 (5R-arugosin M)和5 (Chrysophanol),其中1–4为含异戊烯基的dibenzo[b,e]oxepinone类化合物,5为蒽醌类化合物。1–4对Staphylococcus aureus ATCC43300、ATCC33591,Enterococcus faecium ATCC35667和Bacillussubtilis ATCC6633具有抗菌活性。【结论】含异戊烯基的Dibenzo[b,e]oxepinone类化合物是真菌T. stipitatus中抗菌活性成分。     

猪链球菌2型荚膜唾液酸对细菌毒力和宿主炎症反应的影响

【目的】阐明猪链球菌2型荚膜唾液酸是否影响细菌毒力以及宿主对其炎症反应应答,为研究猪链球菌2型的致病机制奠定基础。【方法】比较实验菌株对BLAB/c小鼠模型的致病性;通过涂板计数的方法检测实验菌株在小鼠体内的分布;观察小鼠脑组织病理改变,分析实验菌株感染小鼠后中枢神经系统的病变差异;从小鼠体外全血细胞水平,运用ELISA法检测实验菌株感染后细胞炎性因子的分泌水平。【结果】荚膜唾液酸合成基因neuB缺失突变株ΔneuB相比野生株05ZYH33株,对小鼠毒力显著降低,回复突变株cΔneuB毒力回复至野生株水平;野生株和突变株在血液及脑组织中分布具有显著差异,均可致BLAB/c小鼠脑组织不同程度的损伤;与野生株组相比较,细菌/细胞相互作用不同时间点后,突变株组体外刺激小鼠全血细胞分泌MCP-1、IL-6的水平显著提高;【结论】荚膜唾液酸影响细菌的毒力及宿主细胞对其的炎症反应应答,它是猪链球菌2型穿透血脑屏障导致脑膜炎的重要毒力因子。     

Expression of the Catalytic Subunit of Ouabain-Sensitive H+,K+-ATPase in Rat Skin Epidermis

A comparative localization of Na⁺,K⁺-ATPase and ouabain-sensitive H⁺,K⁺-ATPase in rat skin was performed using in situ RNA hybridization and immunohistochemistry. Na⁺,K⁺-ATPase was predominantly detected in the basal layer of the epithelium, whereas the ouabain-sensitive H⁺,K⁺-ATPase, in the granular and prickle cell layers. The genes of these ATPases are thus expressed in epithelial cells at different stages of their development. The hypothesis was advanced that the ouabain-sensitive H⁺,K⁺-ATPase is involved in maintaining the skin pH value. The probes specific to the mRNAs of the full-size -subunit of the ouabain-sensitive H⁺,K⁺-ATPase and its truncated form were used to establish a similar distribution of both mRNA variants in skin.     

1H and13C-NMR analysis of four pentasaccharides from urine of blood-group A and B,Leb adults. Confirmation of the structure of two new oligosaccharides: Fuc(α1–2)[GalNAc(α1–3)]Gal(β1–3)[Fuc(α1–4)]Glc and Fuc(α1–2)[Gal(α1–3)]Gal(β1–3)[Fuc(α1–4)]Glc

The major pentasaccharides Fuc(1-2)[GalNAc(1-3)]Gal(1-4)[Fuc(1-3)]Glc and Fuc(1-2) [Gal(1-3)]Gal(1-4)[Fuc(1-3)]Glc, which are normally present in the urine of bloodgroup A Le^b and B Le^b healthy subjects, were each found to be contaminated by a minor component when analysed by¹H-NMR. The determination of these structures, Fuc(1-2) [GalNAc(1-3)]Gal(1-3)[Fuc(1-4)]Glc and Fuc(1-2) [Gal(1-3)]Gal(1-3)[Fuc(1-4)]Glc, was based on the results of methylation analysis and¹H/¹³C-NMR spectroscopy.Abbreviations HPLC high performance liquid chromatography - GLC gas liquid chromatography - NMR nuclear magnetic resonance - COSY correlation spectroscopy - Gal d-galactopyranose - GalNAc 2-acetamido-2-deoxy-d-galactopyranose - Glc d-glucopyranose - Fuc l-fucopyranose - LNDFH I lacto-N-difucohexaose I (Le^b determinant     

Assignment of the1H- and13C-NMR spectra of eight oligosaccharides of the lacto-N-tetraose and neotetraose series

The assignment of the 13C- and 1H-NMR spectra of eight oligosaccharides of the lacto-N-tetraose and neotetraose series was obtained from homonuclear and heteronuclear correlation spectroscopy. These analyses were performed on the following compounds: 1. Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc; 2. NeuAc alpha 2-3Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc; 3. Gal beta 1-3[NeuAc alpha 2-6]GlcNAc beta 1-3Gal beta 1-4Glc; 4. NeuAc alpha 2-3Gal beta 1-3[NeuAc alpha 2-6]GlcNAc beta 1-3Gal beta 1-4Glc; 5. NeuAc alpha 2-3Gal beta 1-3[Fuc alpha 1-4]GlcNAc beta 1-3Gal beta 1-4Glc; 6. Fuc alpha 1-2Gal beta 1-3[NeuAc alpha 2-6]GlcNAc beta 1-3Gal beta 1-4Glc; 7. Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc; 8. NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc.     

(Na + K)-ATPase activity of crude homogenates of rat skeletal muscle as estimated from their K-dependent 3-O-methylfluorescein phosphatase activity

A highly sensitive fluorimetric assay using 3-O-methylfluorescein phosphate as substrate was used in the determination of K⁺-dependent phosphatase activity in preparations of rat skeletal muscle. The gastrocnemius muscle was chosen because of mixed fibre composition. Crude, detergent treated homogenate was used so as to avoid loss of activity during purification. K⁺-dependent phosphatase activities in the range 0.19–0.37 μmol · (g wet weight)⁻¹ · min⁻¹ were obtained, the value decreasing with age and K⁺-deficiency. Complete inhibition of the K⁺-dependent phosphatase was obtained with 10⁻³ M ouabain. Using a KSCN-extracted muscle enzyme the intimate relation between K⁺-dependent phosphatase activity and (Na⁺ + K⁺)-activated ATP hydrolysis could be demonstrated. A molecular activity of 620 min⁻¹ was estimated from simultaneous determination of K⁺-dependent phosphatase activity and [³H]ouabain binding capacity using the partially purified enzyme preparation. The corresponding enzyme concentration in the crude homogenates was calculated and corresponded well with the number of [³H]ouabain binding sites measured in intact muscles or biopsies hereof.     

Elucidation of Structure of Lipid A from the Marine Gram-Negative Bacterium Pseudoalteromonas haloplanktis ATCC 14393T

The chemical structure of lipid A, from the marine -proteobacterium Pseudoalteromonas haloplanktis 14393, a main product of lipopolysaccharide hydrolysis (1% AcOH), was determined using chemical methods and NMR spectroscopy. The lipid A was shown to be -1,6-glucosaminobiose 1,4-diphosphate acylated with two (R)-3-hydroxyalkanoic acid residues at C3 and C3 and amidated with one (R)-3-hydroxydodecanoyl and one (R)-3-dodecanoyloxydodecanoyl residue at N2 and N2, respectively.     

Fluorescent labeling of (Na + K)-ATPase by 5-iodoacetamidofluorescein

5-Iodoacetamidofluorescein (5-IAF) covalently labels dog kidney (Na⁺ + K⁺)-ATPase with approximately 2 moles incorporated per mole of enzyme. ATPase and K⁺-phosphatase activities are fully retained after reaction, and the kinetic parameters for Na⁺, K⁺, Mg²⁺, ATP and p-nitrophenyl phosphate are likewise not significantly affected. The fluorescence of the bound 5-IAF is increased by ATP, Na⁺, and Mg²⁺, and decreased by K⁺. These fluorescence changes likely reflect ligand-induced stabilization of the E₁ or E₂ states of the enzyme.     

Determination of Δ-tetrahydrocannabinol from human saliva by tandem immunoaffinity chromatography-high-performance liquid chromatography

Smoking or ingestion of cannabis causes cognitive, perceptual and behavioural changes, which are responsible for impaired performance in driving motor vehicles. In this paper a novel liquid chromatographic assay for the selective quantification of Δ⁹-tetrahydrocannabinol, the major indicator of a present cannabis intoxication in saliva, is described. The method involves a column-switching procedure and requires an extremely simple pre-treatment of the sample. Deproteinized saliva was directly injected into the chromatographic system. The clean-up and enrichment procedure was performed in an immunoaffinity column, followed by the transfer of the antigens to an octylsilica analytical column. The immunoaffinity sorbent was obtained by covalent immobilization of specific antibodies on epoxy-activated silica. The mobile phase consisted of methanol-aqueous 0.15 mol/1 NaCl solution (elution programmed) and the analyte was detected by measuring the UV absorption at 220 nm. Using an injection volume of 4.5 ml (dilution 3:2, v/v) the limit of quantification was 20 ng/ml, at a signal-to-noise ratio of 5. Recoveries were estimated to be in the range of 70%. Both intra- and inter-day coefficients of variation were below 5%     

Structure of a new nonasaccharide isolated from human milk: VI2Fuc,V4Fuc,III3Fuc-p-lacto-N-hexaose

The structure of a new nonasaccharide isolated from human milk has been investigated. By using methylation analysis, FAB-MS and¹H-and¹³C-NMR spectroscopy as basic methods of structural investigation, this oligosaccharide was identified as VI²--Fuc,V⁴-Fuc,III³--Fuc-p-lacto-n-hexaose: Fuc1-2Gal1-3[Fuc1-4]GlcNAc1-3Gal1-4[Fuc1-3]GlcNAc1-3Gal1-4Glc.Abbreviations COSY correlation spectroscope - DP degree of polymerisation - FAB-MS fast atom bombardment-mass spectrometry - HPLC high performance liquid chromatography - NMR nuclear magnetic resonance - GLC gas-liquid chromatography     

Annular and non-annular binding sites on the (Ca + Mg)-ATPase

Quenching of the fluorescence of the (Ca²⁺ + Mg²⁺)-ATPase purified from muscle sarcoplasmic reticulum can be used to measure relative binding constants of hydrophobic compounds to the phospholipid-protein interface. We show that the binding constant for cholesterol is considerably less than that for phosphatidylcholine, so that cholesterol is effectively excluded from the phospholipid annulus around the ATPase. However, dibromocholestan-3β-ol causes quenching of the fluorescence of the ATPase, and so has access to other, non-annular sites. We suggest that these non-annular sites could be at protein/protein interfaces in ATPase oligomers. Oleic acid can bind at the phospholipid/protein interface, although its binding constant is less than that for a phosphatidylcholine, and it can also bind at the postulated non-annular sites. The effects of these compounds on the activity of the ATPase depend on the structure of the phospholipid present in the systems.     

Nuclear-accumulation kinetics of p9 CksHs1 and p9 CksHs2 in live plant cells correlate with immunochemical characteristics

Summary The two human homologues of the fission yeast cell cycle protein p13 ^suc1 displayed structural characteristics consistent with their existing in solution as differently folded monomers despite 81% identity with respect to their primary structures and both being capable of fulfilling the functions of their homologues in fission and budding yeasts. Carboxyfluorescein-labelled p9 ^CksHs1 and p9 ^CksHs2 retained their native structures. When microinjected into live stamen hair cells ofTradescantia virginiana, the labelled proteins accumulated in the nuclei of the cells. Markedly different nuclearaccumulation kinetics indicated that the human proteins interact differently with other cellular constituents, which supports the proposition that they may have different roles in cellular regulation.Abbreviations Cdk cyclin-dependent kinase - tris tris(hydroxymethyl)aminomethane - Hepes N-(2-hydroxyethyl)piperazine-N-(3-ethanesulphonic acid) - CF 5(6)-carboxyfluorescein-N-hydroxysuccinamide ester - SDS-PAGE sodium dodecyl sulphatepolyacrylamide gel electrophoresis - IEF isoelectric focusing - DEAE Sephacel diethylaminoethyl Sephacel - ELISA enzyme-linked immunosorbent assay - IgG immunoglobulin