Polymorphism of Crystals of Salmonella minnesota Re and Ra Lipopolysaccharides

Salmonella minnesota Re and Ra lipopolysaccharides (LPSs) formed three-dimensional crystals when they were precipitated by the addition of 2 volumes of 95% ethanol containing 375 mM MgCl₂ and incubated in 70% ethanol containing 250 mM MgCl₂ at 4 C. Besides typical shapes of crystals, hexagonal plates and solid columns, which were already reported (J. Bacteriol. 172: 1516–1528 (1990)), the LPSs thus treated formed crystals possessing various shapes such as square or rectangular plate, lozenge plate, discoid, and truncated hexangular pyramid forms. Electron diffraction patterns from all these crystals except square or rectangular plate crystals obtained by electron irradiation from the direction perpendicular to the basal plane were essentially the same as those from hexagonal plate crystals, indicating that they consist of hexagonal lattices with the lattice constant of 4.62 Å. From these results as well as the results of electron microscopic observations of these crystals, it was concluded that all these crystals except square or rectangular plate crystals are composed of hexagonal plate sheets as the basic structural units. Square or rectangular crystals were assumed to correspond to the {1011} planes of solid hexagonal column crystals.     

内毒素诱发鸡多脏器损伤及其对肝TLR4 mRNA表达影响

选取20日龄肉仔鸡随机分为四组,实验组A（灌服LPS100EU／Kg）、B（灌服LPS200EU／Kg）、C（灌服LPS300EU／Kg）和对照组D（灌服生理盐水）。检测不同剂量的LPS在不同时间对鸡多脏器损伤以及内毒素识别因子肝脏Toll样受体4mRNA表达的影响,为进一步揭示禽内毒素血症的发病机理提供依据。实验表明：12～24h是LPS诱发多脏器损伤并引起TLR4表达异常的敏感期,12h肝组织TLR4mRNA的表达水平降至最低,24h后开始回升且随着LPS攻毒剂量的增加,TLR4表达水平回升的速度递减,至48h仍无法达到正常表达水平,这可能与肝细胞作为腺上皮细胞参与TLR4表达调节或机体对抗过度炎症反应而产生的一种保护性反馈引起。     

葡寡糖对LPS诱导小鼠氧化应激与炎症反应调节作用的研究

本文研究了低、中、高三种不同聚合度葡寡糖(LGOS、MGOS、HGOS)对小鼠氧化应激与炎症反应的调节,并探讨其可能的作用机制。在采食正常日粮基础上,各组小鼠每日分别灌胃生理盐水、低聚果糖(FOS)、LGOS、MGOS、HGOS。21 d后,腹腔注射脂多糖(LPS)诱导小鼠建立氧化应激与炎症反应模型,分别测定LPS刺激后4 h和18 h时血清和肝脏中活性氧(reactive oxygen species,ROS)水平、总抗氧化能力(total antioxidant ca-pacity,T-AOC)、过氧化氢酶(catalase,CAT)、谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)活性、丙二醛(Malondialdehyde,MDA)含量、肝脏中一氧化氮(NO)含量、一氧化氮合酶(nitric oxide synthase,NOS)活性、血清中白细胞介素1(Interleukin-1,IL-1)和肿瘤坏死因子(tumor necrosis factor,TNF-a)含量。结果表明:三种聚合度葡寡糖均能显著降低LPS刺激小鼠血清、肝脏中ROS水平(P<0.05),降低肝脏中NO含量、T-NOS、iNOS活性、血清中炎性细胞因子(IL-1、TNF-a)含量(P<0.05),显著提高总抗氧化能力(T-AOC)和抗氧化酶(CAT、GSH-Px)活性(P<0.05)。葡寡糖具有保护机体免受氧化损伤,减缓炎症反应的作用,并随着平均聚合度(degree ofpolymerization,DP)的增加,其调节功能逐渐增强。     

Crystallization and Analyses of Crystals of Various Chemotypes of R-Form Lipopolysaccharides from Salmonella Spp.

Various chemotypes (Re, Rd₂, Rd₁P^–, Rd₁, RcP^–, Rc, Rb₃, Rb₂, Rb₁, and Ra) of R-form lipopolysaccharides (LPSs) of Salmonella spp. were crystallized by treatment with 70% ethanol containing 250 mM MgCl₂, and crystals of the LPSs were observed electron microscopically and analyzed by electron diffraction and synchrotron X-ray diffraction. All the LPSs tested formed three-dimensional crystals showing very similar shapes; hexagonal plate, solid column, discoid, square or rectangular plate, lozenge plate and truncated hexangular or rectangular pyramid forms. Electron diffraction patterns from the hexagonal plate crystals of all these LPSs obtained by electron irradiation from the direction perpendicular to the basal plane showed that they consist of hexagonal lattices with the lattice constant of 4.62 Å. The crystals of all the LPSs thus formed gave ring-like X-ray diffraction patterns because of their small sizes. The long-axis values were calculated from the X-ray diffraction patterns from crystals of all the LPSs in the low-angle region and they corresponded roughly to the length of the proposed primary chemical structures of the R cores of the LPSs. The volume occupied by a single molecule of all the LPSs were calculated from the molecular weights based on the proposed structures and the crystallographic data obtained by electron diffraction, X-ray diffraction, and density determination.     

绿脓杆菌组分疫苗研究进展

由于绿脓杆菌在自然界广泛存在并且对大多数防腐剂和抗菌素药物均表现出耐受性,已成为医院内感染经常出现的条件致病菌。许多年来,人们致力于研制开发安全有效的绿脓杆菌疫苗,以助于对绿脓杆菌感染的控制。目前,一些组分疫苗表现出了较好的发展前景,例如外膜蛋白疫苗、胞外粘液多糖疫苗、LPS疫苗等。本文对这些疫苗的组成成分、制备方法、动物实验和人体观察等作一简要综述。     

LPS、TNFα刺激PBL诱导TRAIL表达的初步研究

近来的实验显示从 EST来源的 TRAIL在体外能特异性地诱导肿瘤细胞凋亡。我们选择人PBL作为人 TRAIL基因的来源 ,抽提细胞在 LPS和 rh- TNFα刺激 2、1 0、1 8h的总 RNA,通过RT- PCR观察 TRAIL基因转录 m RNA的水平 ,然后用 PCR扩增 TRAIL基因 ,并用免疫印迹法分析 TRAIL m RNA翻译蛋白的水平。结果发现 :在 LPS和 rh- TNFα的刺激下可以从 1 0 6 ～ 1 0 7个人 PBL中扩增出 TRAIL基因。提示 LPS和 rh- TNFα刺激细胞并进一步诱导细胞凋亡可能与诱导表达 TRAIL有关。本实验得到了 TRAIL基因 ,为重组表达 TRAIL打下了基础。     

The structure of the antigenic O-polysaccharide of the lipopolysaccharide of Edwardsiella ictaluri strain MT104

The structural characterization of the antigenic O-polysaccharide component of the lipopolysaccharide produced by the fish pathogenic bacterium Edwardsiella ictaluri MT104 was undertaken by the application of NMR spectroscopy and chemical analysis. The O-chain was found to be a linear polymer of a repeating tetrasaccharide unit composed of D-glucose, 2-acetamido-2-deoxy-D-galactose, and D-galactose in a 1:2:1 ratio having the structure: [carbohydrate structure]; see text.     

Crystallization of lipopolysaccharide from a Salmonella typhimurium semi-rough (SR) mutant.

Salmonella typhimurium SR-form lipopolysaccharide (LPS), consisting of a single repeating unit of the O-antigenic polysaccharide, linked to the R-core consisting of oligosaccharide that is, in turn, linked to lipid A, formed crystals whose shapes were hexagonal plates, discoids, and solid columns when precipitated by the addition of 2 volumes of 95% ethanol containing 375 mM MgCl2 and kept in 70% ethanol containing 250 mm MgCl2 at 4 C for 10 days. Among these crystals, the basic form is considered to be the hexagonal plates. Analyses of hexagonal plate crystals showed that they consist of hexagonal lattices with a lattice constant (a axis) of 4.62 A and longitudinal axis (c axis) of approximately 100 A. In X-ray diffraction patterns in the low-angle region, crystals of S. typhimurium SR-form LPS exhibited much less distinct reflections when compared with crystals of synthetic Escherichia coli-type lipid A. In contrast to the previous finding that S. minnesota S-form LPS possessing the O-antigenic polysaccharide does not crystallize under the same experimental conditions as used in the present study, the presence of a single repeating unit of the O-antigenic polysaccharide does not inhibit crystallization.     

The mitogenic principle of Escherichia coli lipoprotein: B-lymphocyte mitogenicity of the synthetic analogue palmitoyl-tetrapeptide (Pam-Ser-Ser-Asn-Ala)

N-Palmitoyl-(S)-seryl-(S)-seryl-(S)-asparaginyl-(S)-alanine (Palmitoyl-tetrapeptide) is an analogue of the N-terminal part of the lipoprotein from the outer membrane of Escherichia coli. It was prepared by chemical synthesis and tested for biological activity in in vitro lymphocyte culture systems. In spleen cells of the inbred mouse strains C3H/HeJ, C3H/He/Bom/ nunu , and Balb/c, the compound exhibited stimulatory activity towards B-lymphocytes comparable to the effect of native lipoprotein, as measured by the incorporation of 3H-thymidine and 3H-uridine, and by a hemolytic plaque assay. The B-lymphocyte tumor cell line BCL1 was also activated by the compound. The results demonstrate, that the N-terminal tetrapeptide moiety of lipoprotein, linked to a lipophilic molecule, constitutes by itself a novel B-lymphocyte mitogen.     

Lysophosphatidylserine dependent incorporation of acyl CoA into phospholipids in rat brain microsomes

[¹⁴C]OleoylCoA was incorporated into phosphatidylinositol 4$\text{1}\text{2}$ times more efficiently than into phosphatidylserine in rat brain and liver microsomes when incubated with various levels of 1-acyl-sn-glycero-3-phosphoserine. In contrast, 1-acyl-sn-glycero-3-phosphocholine dependent incorporation of oleoylCoA was only into phosphatidylcholine. When [l-³H]serine labeled 1-acyl-sn-glycero-3-phosphoserine was used as the labeled substrate, no phosphatidylserine synthesis could be detected in rat brain microsomes. OleoylCoA incorporation in phospholipids in the presence of lysophosphatidylserine was primarily at the 2-position while stearoylCoA was incorporated at the 1-position. These results are interpreted to suggest that there is no acylCoA:1-acyl-sn-glycero-3-phosphoserine acyltransferase in rat brain microsomes and the lysophosphatidylserine dependent position-specific incorporation of acylCoA into various phospholipids may be due to an exchange reaction. A simple highly reproducible one dimensional thin-layer chromatographic system is described for the separation of all the major phospholipids of brain and liver.     

Disintegration of Mg2+ -induced hexagonal assembly of an R-form lipopolysaccharide from Klebsiella pneumoniae by treatment with CaCl2

R-form lipopolysaccharide (LPS) from Klebsiella pneumoniae strain LEN-111 (O3-: K1-), which was precipitated by the addition of 2 volumes of ethanol containing 10 mM MgCl2 for the purification process, ultrastructurally exhibited membrane pieces consisting of an ordered hexagonal lattice structure with a lattice constant of 14 to 15 nm. When the R-form LPS was suspended in 50 mM tris (hydroxymethyl) aminomethane buffer (at pH 8.5) containing 1 mM or higher concentrations of CaCl2 and kept at 4 C for 10 hr, the ordered hexagonal lattice structure of the R-form LPS was disintegrated and changed to an irregular rough, mesh-like structure. By treatment with CaCl2, the content of Mg in the LPS was markedly decreased, and conversely, the content of Ca was increased to a level depending upon the concentration of CaCl2. Results indicate that the addition of CaCl2 to suspensions of the Mg-bound R-form LPS result in a tighter binding of Ca2+ to the R-form LPS and the release of Mg2+ from the R-form LPS, and as a consequence, destroys the Mg2+ -induced ordered hexagonal lattice structure of the R-form LPS.     

鼠抗衣原体属抗原单克隆抗体的研制

本文利用杂交瘤技术,成功地建立了３株稳定分泌小鼠抗衣原体属脂多糖抗原单克隆抗体的杂交瘤细胞。实验结果表明,３株单抗均为ＩｇＭ类,特异性强,识别相似抗原位点,为今后的应用打下了基础。     

腹腔内注射LPS和SEB诱发Ⅰ型IL-1受体在大鼠下丘脑室旁核和视上核的表达增强

目的　为揭示脑内参与神经免疫调节过程的部位和核团。方法　大鼠腹腔内给予细菌内毒素脂多糖 (LPS)或葡萄球菌肠毒素B (SEB) ,用免疫组织化学方法观察了Ⅰ型IL 1受体在脑内表达的变化。结果　Ⅰ型IL 1受体在正常成年大鼠脑内有广泛的表达 ,隔区、视前内侧区、新皮质、海马、下丘脑室旁核、视上核、下丘脑腹内侧核、弓状核和正中隆起等部位有较多Ⅰ型IL 1受体阳性细胞。与生理盐水对照组和非免疫应激对照组 (强迫游泳 )比较 ,LPS或SEB腹腔注射后大鼠下丘脑室旁核和视上核中表达Ⅰ型IL 1受体的细胞数量显著增加 ,染色加深 (P <0 0 5 )。阳性细胞的胞浆染色面积增大 ,突起染色的长度延长。结论　下丘脑室旁核和视上核在神经免疫调节过程中可能具有重要的作用。     

GLN缓解中华鳖免疫应激反应机制的初步研究

本文探讨GLN缓解中华鳖（Trionyxsinensis）免疫应激反应的作用机制,为通过营养调控手段增强机体免疫保护作用,促进水生动物健康养殖提供依据。试验选择体重接近的健康中华鳖60只,随机分为3组,分别腹腔注射0．7％NaCl、500μgLPS／KgBW、500μgLPS＋100mgGLN／KgBW。检测不同时段血浆COR、TNF—α、IL-1β、IL-6、IgM、IgG、IgA、LSZ、GSH、GSH—PX、MDA以及肝脏PRO、GSH、GSH—PX、MDA含量的变化。结果表明：注射LPS引起中华鳖产生了免疫应激反应,血浆中COR、TNF—α、IL-1β、IL-6、IgM、GSH—PX、MDA和肝脏组织PRO、GSH—PX、MDA含量在相应时段显著升高（P〈0．05）,而血浆和肝脏GSH含量显著降低（P〈0．05）;补充GLN不仅缓解了COR、TNF—α、IL-1β和IL-6等的上升趋势,而且使非特异性免疫抑制剂（MDA）水平降低。LPS作为免疫原可引起中华鳖产生免疫应激反应,补充GLN可以缓解免疫应激,对中华鳖具有免疫保护作用。     

Occurence of antibodies against chlamydial lipopolysaccharide in human sera as measured by ELISA using an artificial glycoconjugate antigen

Abstract An artificial glycoconjugate containing, as a ligand, the deacylated carbohydrate backbone of a recombinant Chlamydia -specific lipopolysaccharide was used as a solid-phase antigen in ELISA to measure antibodies against chlamydial LPS. The specificity and reproducibility of the assay was shown by using a panel of prototype monoclonal antibodies representing the spectrum of antibodies also occuring in patient sera. These mAbs recognized Chlamydia -specific epitopes [ α 2→8-linked disaccharide of 3-deoxy- d - manno -octulosonic acid (Kdo) or the trisaccharide α Kdo-(2→8)-→Kdo] or those shared between chlamydial and Re-type LPS ( α Kdo, α →4-linked Kdo disacccharide). The assay was used to measure IgG, IgA and IgM antibodies against chlamydial LPS in patients with genital or respiratory tract infections. In comparison to the results obtained with sera from blood donors, it became evident that both types of infection result in significant changes in the profile of LPS antibodies.     

Protective effect of OK-432 on mice against endotoxemia and infection with Pseudomonas aeruginosa and Salmonella enteritidis

OK-432 has been used clinically as a biological response modifier for cancer therapy. We investigated here the protective effects of OK-432 against endotoxic shock and infectious death caused by Pseudomonas aeruginosa and Salmonella enteritidis in mice and proposed a possible mechanism. Pretreatment of OK-432 reduced the lethality of lipopolysaccharide (LPS)-induced endotoxic shock in D-(+)-galactosamine-sensitized C3H/HeN mice. OK-432 did not affect the TNFalpha production in blood, but it did decrease the susceptibility to TNFalpha. Furthermore, an acceleration of LPS clearance from blood was detected. The pretreatment of OK-432 also decreased the lethality of mice in bacterial infection caused by P. aeruginosa and S. enteritidis. The rapid decrease of the viable bacteria from the circulating blood and in spleen and liver in mice was observed in a manner similar to LPS clearance. These findings indicate that the protective effect of OK-432 against the endotoxemia and bacteremia may depend on an up-regulation of clearance of LPS and bacteria and the augmented resistance to TNFalpha.     

抗炎多肽AF-2对LPS诱导的小鼠急性肺损伤的保护

目的研究抗炎多肽AF-2（antiflammin-2）对内毒素（LPS）诱导的小鼠急性肺损伤的保护作用。方法Balb/c雄性小鼠37只,随机分为3组,对照组（n=10）、急性肺损伤（ALI）模型组（n=14）和AF-2治疗组（n=13）,模型组和治疗组腹腔注射大肠杆菌内毒素复制小鼠肺损伤模型,治疗组同时注射抗炎多肽AF-2,对照组和模型组注射等量的生理盐水。在0h、6h和12h记录动物的呼吸频率,12h处死动物,肺组织切片观察肺病理变化,ELISA法检测血清细胞因子。结果6h和12hAF-2治疗组动物呼吸频率均低于模型组。肺组织病理显示AF-2对LPS诱导的小鼠ALI肺组织的渗出、炎细胞浸润有一定的抑制作用。AF-2治疗组与ALI模型组比较血清IL-6水平明显下降。结论AF-2对内毒素诱导的小鼠急性肺损伤有一定的保护作用。     

Serum antibodies of periodontitis patients compared to the lipopolysaccharides of Porphyromonas gingivalis and Fusobacterium nucleatum

Serum antibody titers against the lipopolysaccharides (LPSs) of Porphyromonas gingivalis and Fusobacterium nucleatum were compared between 9 periodontitis patients and 24 healthy persons. The IgG titers against the LPSs of P. gingivalis ATCC 33277(T) and W50 were clearly higher in the patients than in the healthy persons. However, IgM titers against the LPSs of P. gingivalis strains were relatively low, and no significant difference was observed between the patients and healthy persons. On the other hand, IgG and IgM titers against the LPS of Fusobacterium nucleatum JCM 8532(T) in some patients were significantly higher than those in the healthy persons, although the difference in IgG titers was not large compared to that of the LPS of P. gingivalis. These results suggest that the antibody measurement of patients' sera against the LPS of periodontal bacteria can be applied for the diagnosis of periodontitis.     

O139霍乱弧菌LPS基因在大肠杆菌中的克隆和表达

利用粘粒载体pCOS5构建了国内分离的O139霍乱弧菌的基因组文库,并从文库中筛选获得可以表达O139霍乱弧菌脂多糖的重组克隆株E.coliJM109(pMG310)。重组粘粒pMG310经酶切分析,所克隆的外源DNA片段大小为37kb。实验证明：重组克隆株E.coliJM109(pMG310)所表达的脂多糖具有良好的免疫原性及反应原性。