Characterization of Neospora caninum macrophage migration inhibitory factor

The present study is the first characterization of Neospora caninum macrophage migration inhibitory factor (NcMIF). BLAST-N analysis of NcMIF revealed high similarity (87%) to the Toxoplasma gondii MIF. NcMIF was cloned and expressed in Escherichia coli in 3 forms, NcMIF (mature protein), NcMIFm (mutation of proline-2 to glycine), and NcMIFhis (addition of a polyhistidine tag at the N-terminus). None of these recombinant NcMIFs (rNcMIF) had tautomerase, oxidoreductase, or immunologic regulatory activities. rNcMIF was unable to compete with recombinant human MIF for a MIF receptor (CD74), suggesting that NcMIF does not bind to this MIF receptor. The glycine substitution for proline-2 of NcMIF resulted in increased retention time on SEC-HPLC and decreased formation of dimers and trimers. The addition of N-terminal HIS-tag led to increased formation of trimers. Immunofluorescence staining demonstrated that NcMIF was localized to the apical end of N. caninum tachyzoites. Immunoelectron microscopy further revealed that NcMIF was present in the micronemes, rhoptries, dense granules, and nuclei. NcMIF was abundant in the tachyzoite lysate and present in excretory and secretory antigen (ESAg) preparations. Total and secretory NcMIF was more abundant in a non-pathologic clone, Ncts-8, than in the wild type isolate (NC1). Furthermore, NcMIF release by the both isolates was increased in the presence of calcium ionophore. This differential production of NcMIF by the pathologic and non-pathologic isolates of N. caninum may suggest a critical role of this molecule in the infectious pathogenesis of this parasite.     

呼吸窘迫综合症患儿外周血单个核细胞中CD24和炎症因子mRNA的表达水平及其预后价值

目的:通过检查呼吸窘迫综合症患儿外周血单个核细胞中CD24和TNF-α、IL-6、和IL-17A炎症因子mRNA的表达,探讨其对呼吸窘迫综合症的诊断和预后价值。方法:选择2015年1月至12月在我院接受治疗的32例非感染型呼吸窘迫综合症患儿为研究组,选择同期的30例健康新生患儿为对照组,采集研究组治疗前后和对照组的外周血,分离单个核细胞,采用RT-PCR检测CD24和TNF-α、IL-6、和IL-17A炎症因子mRNA的表达水平。结果:研究组治疗前CD24mRNA表达水平明显高于对照组,差异有统计学意义(P0.01),而TNF-α、IL-6、和IL-17A mRNA表达水平比较,差异无统计学意义(P0.05)。研究组治疗后CD24mRNA表达水平明显低于治疗前,差异有统计学意义(P0.01),而TNF-α、IL-6、和IL-17A mRNA表达水平比较,差异无统计学意义(P0.05)。结论:呼吸窘迫综合症患儿外周血单个核细胞中CD24mRNA表达水平升高,可能是其诊断和预后的分子标记物。     

Receptins: a novel term for an expanding spectrum of natural and engineered microbial proteins with binding properties for mammalian proteins

A new term ‘receptin’, derived from recipere (lat.), is proposed to denote microbial binding proteins that interact with mammalian target proteins. An example of such a ‘receptin’ is staphyloccocal protein A which binds to the Fc part of many mammalian immunoglobulins. Several other types of ‘receptins’ are listed. This term may easily be distinguished from the similar term ‘receptor’, describing a binding site on a cell surface, mostly eukaryotic, where a secondary effect is induced inside the cell upon binding to a ligand. A receptin, however, does not necessarily have to induce a secondary event. Receptins include so called MSCRAMMs, adhesins, and also engineered receptins, affibodies, and engineered ligands. It denotes any protein of microbial origin, cell‐bound or soluble, which can bind to a mammalian protein. It fulfills the need for an umbrella terminology for a large group of binding structures. In contrast, the term ‘lectin’ represents a group of proteins with affinity for carbohydrate structures. The new term ‘receptin’ includes a number of key microbial proteins involved in host–parasite interactions and in virulence. Some receptins are promising vaccine candidates. Copyright © 1999 John Wiley & Sons, Ltd.     

Number of receptor sites from Scatchard and Klotz graphs: complementary approaches

Estimates of number of receptor sites and evaluation of the complexity of the binding process require collection of a spectrum of binding measurements and selection of a theoretical model to fit the experimental data. The appropriateness of the measurements and of the model can be visually judged on graphic displays of the model-data fitting curves in Scatchard and semilogarithmic coordinates. This approach is helpful for detecting the two types of errors most frequently found in reports of binding studies: (1) underestimating the number of binding sites, and (2) failure to recognize the complexity of the binding process. While the former is readily recognizable on semilogarithmic but not on Scatchard plots of the model fitting the data, the latter might not be apparent on either plot. Collection of extensive measurements over a wide range of ligand concentrations with graphic display of the model-data fitting curves in Scatchard and semilogarithmic coordinates should be used to recognize and prevent both errors.     

Polarized expression of CD74 by gastric epithelial cells.

CD74 is known as the major histocompatibility complex (MHC) class II-associated invariant chain (Ii) that regulates the cell biology and functions of MHC class II molecules. Class II MHC and Ii expression was believed to be restricted to classical antigen-presenting cells (APC); however, during inflammation, other cell types, including mucosal epithelial cells, have also been reported to express class II MHC molecules. Given the importance of Ii in the biology of class II MHC, we sought to examine the expression of Ii by gastric epithelial cells (GEC) to determine whether class II MHC molecules in these nonconventional APC cells were under the control of Ii and to further support the role that these cells may play in local immune and inflammatory responses during Helicobacter pylori infection. Thus we examined the expression of Ii on GEC from human biopsy samples and then confirmed this observation using independent methods on several GEC lines. The mRNA for Ii was detected by RT-PCR, and the various protein isoforms were also detected. Interestingly, these cells have a high level expression of surface Ii, which is polarized to the apical surface. These studies are the first to demonstrate the constitutive expression of Ii by human GEC.     

冠心病患者血浆CD69、DKK-1与血脂、炎性因子的关系及其诊断价值分析

摘要 目的：分析冠心病（CHD）患者血浆CD69、Wnt拮抗剂蛋白-1（DKK-1）与血脂、炎性因子的关系,并分析CD69和DKK1对于CHD患者的诊断价值。方法：选取2016年6月~2018年12月在我院诊治的CHD患者136例,根据Gensini评分分为轻度亚组、中度亚组、重度亚组,同时纳入与之年龄、性别匹配的冠状动脉造影正常者136例作为对照组。检测受试者血清炎性因子、血脂、及血浆CD69、DKK-1水平,分析CHD患者CD69、DKK-1水平与血脂、炎性因子、Gensini评分的相关性,采用受试者工作特征（ROC）曲线分析CD69和DKK1对于CHD患者的诊断价值。结果：CHD患者CD69、DKK-1、白细胞介素-10（IL-10)、白细胞介素-6（IL-6）、三酰甘油（TG）、总胆固醇（TC）、低密度脂蛋白（LDL）水平高于对照组,高密度脂蛋白（HDL）水平低于对照组（P<0.05）;随着冠脉狭窄病变程度的加重,CD69、DKK-1、IL-10、IL-6、TC、TG、LDL水平呈升高趋势,HDL水平呈降低趋势（P<0.05）。CHD患者CD69、DKK-1水平与IL-10、IL-6、TC、TG、LDL、Gensini评分均呈正相关,与HDL呈负相关（P<0.05）。ROC曲线分析结果显示,CD69、DKK-1联合检测诊断CHD的灵敏度、特异度分别为0.816、0.846。结论：CHD患者血浆DKK-1、CD69水平升高,与血脂、炎性因子和Gensini评分密切相关,两者联合检测可提高CHD诊断效能。     

Ligand-receptor interactions: detailed evaluation of occupancy-dependent affinity

The exact nature of the curvilinearity of Scatchard plots derived from hormonal and nonhormonal binding systems has not been definitively resolved. Such plots are compatible with heterogeneous receptors with different but fixed affinities and with negatively interacting binding sites resulting in occupancy-dependent affinity. In the current study we examined in detail the effect of receptor occupancy by the ligand on receptor affinity under a variety of experimental conditions. We chose the human lymphocyte-leukoagglutinin (LPHA) system, which closely mimics the IM9-insulin model. Reliable estimates of total binding capacity (728 ng/10(6) cells) essential to our report were calculated from a wide database by the least-squares model. At occupancies greater than or equal to 0.085, receptors are associated with low and fixed affinity (1.5 X 10(6) M-1), whereas at occupancies less than or equal to 0.085, affinity is high and fixed (1.8 X 10(8) M-1) or high but variable (1 X 10(7) M-1 to 1.5 X 10(6) M-1) depending on whether the binding is assumed to be noncooperative or cooperative, respectively. Calculation of receptor-ligand complex dissociation velocity over a wide range of occupancies (0.01-0.40) suggested that occupancy exerts an inversely proportional effect on affinity that is rapid and sustained. Cell activation (DNA synthesis) is initiated at receptor occupancy of approximately equal to 0.004 and is magnified as ligand binding to high affinity receptors increases up to approximately equal to 0.07 occupancy (functional sites), beyond which point further binding (to low affinity sites) becomes increasingly ineffective and cytotoxic (redundant sites). These findings suggest that occupancy influences affinity as postulated by the hypothesis of negative cooperativity. Through this effect occupancy may play a significant role in regulating ligand-induced cell responses.     

Estrogen-inducible uterine flavonoid binding sites: is it time to reconsider?

Epidemiological data support the beneficial effect of plant flavonoids on human health including anti-inflammatory and cancer preventing actions. The phytoestrogen flavonoids might interfere with estrogen action. The possible relations between the steroid- and the flavonoid-signalling in animal and plant cells have been addressed in numerous studies in the past decade. In search for possible sites of conjunction between these phenomena the post-receptor targets must not be disregarded.

The estrogen-inducible type II estrogen binding sites of rat uteri have first been reported 25 years ago by Clark and coworkers [Biochem. Biophys. Res. Commun. 81 (1978) 1]. These sites are known to bind catecholic flavonoids with considerable affinity. Behaviour of the tyrosinase-like enzymatic activity associated with these sites appeared reminiscent to the recently described dopachrome oxidase or tautomerase activity exhibited by the cytokine macrophage migration inhibitory factor (MIF) inasmuch as it also accepts a broad range of catecholic melanogenic precursors. Therefore we assessed, whether the known type II ligand flavonoids interfere with the MIF tautomerase. We report here, that luteolin and quercetin have a biphasic effect on the enol–keto conversion of phenylpyruvate mediated by MIF tautomerase. We also demonstrate the presence of MIF immunoreactivity by Western blotting in rat uterine nuclear extracts prepared according to the method that yields high type II binding activity. These data support the possible participation of MIF in type II estrogen binding phenomena.     

Structural segments and residue propensities in protein-RNA interfaces: comparison with protein-protein and protein-DNA complexes

The interface of a protein molecule that is involved in binding another protein, DNA or RNA has been characterized in terms of the number of unique secondary structural segments (SSSs), made up of stretches of helix, strand and non-regular (NR) regions. On average 10-11 segments define the protein interface in protein-protein (PP) and protein-DNA (PD) complexes, while the number is higher (14) for protein-RNA (PR) complexes. While the length of helical segments in PP interaction increases with the interface area, this is not the case in PD and PR complexes. The propensities of residues to occur in the three types of secondary structural elements (SSEs) in the interface relative to the corresponding elements in the protein tertiary structures have been calculated. Arg, Lys, Asn, Tyr, His and Gln are preferred residues in PR complexes; in addition, Ser and Thr are also favoured in PD interfaces.     

胰胆管造影术后发生胰腺炎的危险因素分析

目的:探讨经胰胆管造影术后患者发生胰腺炎的危险因素,以期为临床实践提供一定的参考依据。方法:回顾性分析2015年1月1日至2018年1月1日入住我院行胰胆管造影术的患者,根据纳入排除标准,分成胰腺炎组和非胰腺炎组,比较分析两组患者的性别、年龄、既往行内镜逆行性胰胆管造影术(endoscopic retrograde pancreatic cholangiography,ERCP)与否、ERCP次数、ERCP手术时间、胆道括约肌气囊扩张术、插管困难等因素和血清炎症因子TNF-α、IL-1、IL-6、IL-10的表达情况。结果:研究共纳入1891例患者,并发胰腺炎者124例(6.55%),非胰腺炎患者1767例。性别、年龄、既往行ERCP、ERCP次数、ERCP手术时间、胆道括约肌气囊扩张术、插管困难均为胰腺炎发生的危险因素,且ERCP手术时间和胆道括约肌气囊扩张术是为并发胰腺炎的独立危险因素。ERCP术后胰腺炎组血清TNF-α、IL-1、IL-6、IL-10的表达水平均显著高于非胰腺炎组(P0.05)。结论:ERCP手术时间、胆道括约肌气囊扩张术及高血清炎症因子水平可能是并发胰腺炎的独立危险因素。     

Modulating calmodulin binding specificity through computational protein design

We report the computational redesign of the protein-binding interface of calmodulin (CaM), a small, ubiquitous Ca(2+)-binding protein that is known to bind to and regulate a variety of functionally and structurally diverse proteins. The CaM binding interface was optimized to improve binding specificity towards one of its natural targets, smooth muscle myosin light chain kinase (smMLCK). The optimization was performed using optimization of rotamers by iterative techniques (ORBIT), a protein design program that utilizes a physically based force-field and the Dead-End Elimination theorem to compute sequences that are optimal for a given protein scaffold. Starting from the structure of the CaM-smMLCK complex, the program considered 10(22) amino acid residue sequences to obtain the lowest-energy CaM sequence. The resulting eightfold mutant, CaM_8, was constructed and tested for binding to a set of seven CaM target peptides. CaM_8 displayed high binding affinity to the smMLCK peptide (1.3nM), similar to that of the wild-type protein (1.8nM). The affinity of CaM_8 to six other target peptides was reduced, as intended, by 1.5-fold to 86-fold. Hence, CaM_8 exhibited increased binding specificity, preferring the smMLCK peptide to the other targets. Studies of this type may increase our understanding of the origins of binding specificity in protein-ligand complexes and may provide valuable information that can be used in the design of novel protein receptors and/or ligands.     

单纯性肥胖儿童血清Hcy、visfatin、E-FABP水平与糖脂代谢紊乱和炎症因子的相关性分析

摘要 目的：研究单纯性肥胖儿童血清同型半胱氨酸（Hcy）、内脂素（visfatin）、上皮型脂肪酸结合蛋白（E-FABP）水平与糖脂代谢紊乱和炎症因子的相关性。方法：将2019年4月～2020年10月于我院就诊的70例单纯性肥胖儿童纳入研究,记作肥胖组。另取同期于我院接受体检的健康儿童70例作为对照组。检测并比较两组血清Hcy、visfatin、E-FABP水平,糖脂代谢紊乱相关指标和炎症因子水平。以Pearson相关性分析明确单纯性肥胖儿童血清Hcy、visfatin、E-FABP水平与糖脂代谢紊乱和炎症因子的关系。结果：肥胖组血清Hcy、visfatin、E-FABP水平均高于对照组（均P＜0.05）。肥胖组空腹血糖（FPG）、空腹胰岛素（FINS）水平及胰岛素抵抗指数（HOMA-IR）均高于对照组（均P＜0.05）。肥胖组总胆固醇（TC）、甘油三酯（TG）、低密度脂蛋白胆固醇（LDL-C）水平均高于对照组,而高密度脂蛋白胆固醇（HDL-C）水平低于对照组（均P＜0.05）。肥胖组血清白细胞介素-1β（IL-1β）、白细胞介素-6（IL-6）及肿瘤坏死因子-α（TNF-α）水平均高于对照组（均P＜0.05）。经Pearson相关性分析可得：单纯性肥胖儿童血清Hcy、visfatin、E-FABP水平与FPG、FINS、HOMA-IR、TC、TG、LDL-C、IL-1β、IL-6、TNF-α水平均呈正相关,而与HDL-C水平呈负相关（均P＜0.05）。结论：单纯性肥胖儿童血清Hcy、visfatin、E-FABP水平均异常升高,且与其糖脂代谢紊乱及炎症反应密切相关,值得临床重点关注。     

Identification of protein interaction partners and protein-protein interaction sites

Rigid-body docking has become quite successful in predicting the correct conformations of binary protein complexes, at least when the constituent proteins do not undergo large conformational changes upon binding. However, determining whether two given proteins interact is a more difficult problem. Successful docking procedures often give equally good scores for proteins that do not interact experimentally. This is the case for the multiple minimization approach we use here. An analysis of the results where all proteins within a set are docked with all other proteins (complete cross-docking) shows that the predictions can be greatly improved if the location of the correct binding interface on each protein is known, since the experimental complexes are much more likely to bring these two interfaces into contact, at the same time as yielding good interaction energy scores. While various methods exist for identifying binding interfaces, it is shown that simply studying the interaction of all potential protein pairs within a data set can itself help to identify the correct interfaces.