蜂毒素与紫膜的相互作用机理研究──与三种表面活性剂的比较

用吸收光谱和圆二色谱的方法研究了蜂毒素与嗜血菌紫膜的相互作用机理．通过与三种在结构和电荷上不同的表面活性剂与紫膜的作用相比较,可以年出蜂毒作为带正电荷的分子与同样带正电的表面活性剂ＤＴＡＢ在引起紫膜凝聚方面表现相同;但在对紫膜可见光区的吸收光谱和圆二色谱的影响上却与具有刚性结构的ＣＨＡＰＳ相似,表明蜂毒可在紫膜表面以一种刚性较大的构象（如α螺旋）存在,不能进入膜蛋白流水区的深层．另外,从紫膜－Ｔｒｉｔｏｎ－蜂毒混合作用体系的研究中得到如下推测：蜂毒与Ｔｒｉｔｏｎ竞争菌紫质分子周围的结合位点,可排斥位于菌紫质周围的Ｔｒｉｔｏｎ分子．表明蜂毒具有比Ｔｒｉｔｏｎ更强的与菌紫质的亲和力,从而提供了支持蜂毒分子存在与膜蛋白－菌紫质的直接相互作用的有力证据．     

The disulfide bridges of the immunoglobulin-like domains of FcγRIIIB are essential for efficient expression and biological activity

The immunoglobulin G receptor FcRIIIB belongs to the immunoglobulin superfamily as two extracellular domains show homology to the immunoglobulin domains. Since some residues in these domains, such as the two cysteines, are supposed to form an intrachain disulfide bridge are so commonly conserved, they may be of importance for correct folding. Site-directed mutagenesis and expression in BHK21 confirmed this supposition for the FcRIIIB. Replacing both cysteines in the first and/or second domain by serines reduced the surface expression level by 50%, whereas the ligand binding capability was 20–30% of that seen in cells expressing the wild-type receptor. Replacing one of the four cysteines resulted in the loss of surface expression. Exchanging the conserved tryptophan in the first domain by phenylalanine only slightly affected the ligand binding (25%), whereas the surface expression remained unchanged.     

Hectogram-scale synthesis of [Aib8, Arg34]-GLP-1 (7–37) by liquid-phase fragment condensation

Based on small-scale synthesis (0.3 g), a 100-g scale-up synthesis of crude [Aib⁸, Arg³⁴]-glucagon-like peptide-1 (GLP-1) (7–37) was completed. The crude [Aib⁸, Arg³⁴]-GLP-1 (7–37) was purified using a dynamic axial compression column 200 (DAC-200). Approximately 61 g of [Aib⁸, Arg³⁴]-GLP-1 (7–37) with a purity of >99% was obtained through one-step reverse-phase chromatography. The purification yield was approximately 92%. The yield from the total reaction was approximately 60%. In summary, we developed an economical and environmentally friendly route to the synthesis and purification of crude [Aib⁸, Arg³⁴]-GLP-1 (7–37), laying a foundation for subsequent industrial production.     

UreB immunodominant epitope-specific CD8+ T-cell responses were beneficial in reducing gastric symptoms in Helicobacter pylori-infected individuals

Background and aims

Although Helicobacter pylori is recognized as an extracellular infection bacterium, it can lead to an increase in the number of CD8⁺ T cells after infection. At present, the characteristics of H. pylori antigen-specific CD8⁺ T cells and the epitope response have not been elucidated. This study was focused on putative protective antigen UreB to detect specific CD8⁺ T-cell responses in vitro and screen for predominant response epitopes.

Methods

The PBMCs collected from H. pylori-infected individuals were stimulated by UreB peptide pools in vitro to identify the immunodominant CD8⁺ T-cell epitopes. Furthermore, their HLA restriction characteristics were detected accordingly by NGS. Finally, the relationship between immunodominant responses and appearance of gastric symptoms after H. pylori infection was conducted.

Results

UreB-specific CD8⁺ T-cell responses were detected in H. pylori-infected individuals. Three of UreB dominant epitopes (A-2 (UreB_443–451: GVKPNMIIK), B-4 (UreB_420–428: SEYVGSVEV), and C-1 (UreB_5–13: SRKEYVSMY)) were firstly identified and mainly presented by HLA-A*1101, HLA-B*4001 and HLA-C*0702 alleles, respectively. C-1 responses were mostly occurred in H. pylori-infected subjects without gastric symptoms and may alleviate the degree of gastric inflammation.

Conclusions

The UreB dominant epitope-specific CD8⁺ T-cell response was closely related to the gastric symptoms after H. pylori infection, and the C-1 (UreB_5-13) dominant peptides may be protective epitopes.     

In vitro and in vivo properties of an anti-CD5—momordin immunotoxin on normal and neoplastic T lymphocytes

An anti-CD5 monoclonal antibody (mAb) was linked to the plant toxin momordin, a type-1 ribosome-inactivating protein purified fromMomordica charantia. The in vitro cytotoxicity of the immunotoxin was evaluated as the inhibition of protein and/or DNA synthesis on isolated peripheral blood mononuclear cells (PBMC) and on human T cell leukemia Jurkat. The potency of the immunotoxin on PBMC was very high (IC₅₀ = 1–10 pM) and was not affected by blood components. The conjugate was also very efficient in the inhibition of the proliferative response in a mixed lymphocyte reaction (IC₅₀ = 10 pM). Moreover, the in vitro performances of the immunotoxin compared favourably with those reported for other anti-CD5-based immunoconjugates containing ricin A chain. The in vivo activity of the immunotoxin was assessed in the model ofnu/nu mice bearing Jurkat leukemia. A significant inhibition of the tumour development (80%,P <0.01) in the animals treated with immunotoxin was observed. Taken together, the in vitro and in vivo results suggest that the anti-CD5-momordin conjugate may be useful for graft-versus-host disease therapy and potentially in the treatment of CD5-positive leukemias and lymphomas.     

A Glycoprotein Expressed by Human Fibrous Astrocytes is a Hyaluronate-Binding Protein and a Member of the CD44 Family

We have isolated and characterized an antigen from normal human brain called p80, so called because it migrated with an Mr of 80 kDa on SDS PAGE. The Mr of 80 kDa consists of a protein of about 55-60 kDa and carbohydrate (20-25 kDa). The carbohydrate is almost entirely of the N-linked type, although a small amount of O-linked carbohydrate was detected. Cross-reactivity with monoclonal antibodies A3D8 and A1G3 showed that p80 could therefore be considered an isoform of the CD44 adhesion molecules. In addition, specific binding to hyaluronate which was not competed for by proteoglycan demonstrated that it involved different sites than the proteoglycan binding sites. We also observed that fucoidan and dextran sulphate increased the binding by 200-250% while chondroitin sulphate C also increased the binding but to a lesser extent. Heparin, heparan sulphate and chondroitin sulphates A and B did not have such an effect. The binding of p80 to hyaluronate was pH dependent with a maximum at pH 6.4. We concluded that p80 was an astrocyte specific adhesion molecule.     

Biosynthesis and function of membrane bound and secreted forms of recombinant CD11b/CD18 (Mac-1)

Full-length (membrane bound) and truncated (secreted) forms of the beta 2 integrin heterodimer, CD11b/CD18 (Mac-1), were expressed in a human kidney cell line (293) that normally does not express leukocyte adhesion molecules (Leu-CAMs). The biosynthesis of recombinant Mac-1 in 293 cells differed from that reported for leukocytes in that heterodimer formation was not required for CD11b to be exported to the cell surface. A stable cell line was constructed that constitutively secreted the recombinant, truncated Mac-1 heterodimer into growth conditioned cell culture medium. A novel monoclonal antibody that enabled an immunoaffinity method for the selective purification of recombinant Mac-1 heterodimers was identified. Sufficient protein was purified to allow the first measurement of the 50% inhibitory concentration (IC₅₀) for CD11b/CD18 and for the direct comparison of the inhibitory activity of recombinant soluble Mac-1 with that of various CD18 and CD11b specific monoclonal antibodies. Purified recombinant soluble Mac-1 inhibited the binding of neutrophils, activated by opsonized zymosan or fMet-Leu-Phe peptide, to human umbilical vein endothelial cells. Similarly, the recombinant integrin was effective in inhibiting the binding of unactivated neutrophils to tumor necrosis factor (TNF-alpha) activated endothelial cells. The availability of an abundant source of purified, biologically active Mac-1 will enable direct physical and chemical investigations into the relationship between the structure and function of this leukocyte adhesion molecule.     

传染性单核细胞增多症患儿NLR、CD4+/CD8+比值、ADA与EBV-DNA载量的相关性分析及对肝损害的影响

摘要 目的：探讨传染性单核细胞增多症（IM）患儿外周血中性粒细胞/淋巴细胞比值（NLR）、CD4⁺/CD8⁺比值、腺苷脱氨酶（ADA）与EB病毒（EBV）-脱氧核糖核酸（DNA）载量的相关性,分析其对IM患儿肝损害的影响。方法：选择2019年1月至2022年4月我院儿科收治的102例IM患儿（IM组）,另选择同期我科收治的95例EB病毒检测阴性的发热患儿（非IM组）和体检健康的73例健康儿童（对照组）。根据是否发生肝损害将IM患儿分为肝损害组（61例）和非肝损害组（41例）。比较外周血NLR、CD4⁺/CD8⁺比值、ADA与EBV-DNA载量,Pearson法分析NLR、CD4⁺/CD8⁺比值、ADA与EBV-DNA载量的相关性。多因素Logistic回归分析IM患儿发生肝损害的影响因素。结果：IM组ADA高于非IM组和对照组（P＜0.05）,且非IM组高于对照组（P＜0.05）,NLR、CD4⁺/CD8⁺比值低于非IM组和对照组（P＜0.05）,且非IM组低于对照组（P＜0.05）,IM组EBV-DNA载量高于非IM组（P＜0.05）。IM患儿ADA与EBV-DNA载量呈正相关（r=0.493,P＜0.05）,NLR、CD4⁺/CD8⁺比值与EBV-DNA载量呈负相关（r=-0.419、-472,P＜0.05）。肝损害组ADA、EBV-DNA载量高于非肝损害组（P＜0.05）,NLR、CD4⁺/CD8+⁺比值低于非肝损害组（P＜0.05）。肝脏肿大、高EBV-DNA载量、高ADA是IM患儿肝损害的危险因素（P＜0.05）,高NLR、高CD4⁺/ CD8⁺比值是保护因素（P＜0.05）。结论：IM患儿ADA增高,NLR、CD4⁺/CD8⁺比值降低,与EBV-DNA载量增加以及肝损害有关。     

宫颈癌组织miR-200b-5p、miR-424-5p表达与临床病理特征、PI3K/AKT/mTOR信号通路和预后的关系研究

摘要 目的：探讨宫颈癌组织微小核糖核酸（miRNA）-200b-5p、miR-424-5p表达与临床病理特征、磷脂酰肌醇3-激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白（PI3K/AKT/mTOR）信号通路和预后的关系。方法：选取2016年7月～2019年6月西安市中心医院收治的123例宫颈癌患者,采用定量聚合酶链式反应（qPCR）检测癌组织与癌旁组织中miR-200b-5p、miR-424-5p、PI3K信使RNA（mRNA）、AKT mRNA、mTOR mRNA表达。分析miR-200b-5p、miR-424-5p表达与PI3K mRNA、AKT mRNA、mTOR mRNA表达的相关性及与临床病理特征的关系。采用K-M法绘制不同miR-200b-5p、miR-424-5p表达宫颈癌患者生存曲线。结果：与癌旁组织比较,宫颈癌组织中miR-200b-5p、miR-424-5p表达降低,PI3K mRNA、AKT mRNA、mTOR mRNA表达升高（P＜0.05）。Pearson相关性分析显示,宫颈癌组织中miR-200b-5p、miR-424-5p表达与PI3K mRNA、AKT mRNA、mTOR mRNA表达均呈负相关,PI3K mRNA、AKT mRNA、mTOR mRNA表达呈两两相关（P＜0.05）。miR-200b-5p、miR-424-5p表达与宫颈癌分化程度、国际妇产科联盟（FIGO）分期和淋巴结转移有关（P＜0.05）。随访3年,123例宫颈癌患者累积生存率为62.60%（77/123）。K-M生存曲线分析显示,miR-200b-5p、miR-424-5p高表达组累积生存率分别高于miR-200b-5p、miR-424-5p低表达组（P＜0.05）。结论：宫颈癌组织中miR-200b-5p、miR-424-5p低表达,与分化程度、FIGO分期、淋巴结转移、PI3K/AKT/mTOR信号通路和预后有关。