Exploring Pleiotropic Functions of Canine β-Defensin 103: Nasal Cavity Expression,Antimicrobial Activity,and Melanocortin Receptor Activity

Canine β-defensin 103 (cBD103) and its common variant cBD103ΔG23 are multitasking polypeptides. As a β-defensin, cBD103 is one of many antimicrobial agents used by the innate immunity to thwart pathogenic colonization. In this study, we showed that cBD103 was expressed throughout the nasal cavity, with primary expression in the nares as well as respiratory and olfactory epithelia. In the rostral nasal concha, cBD103 was expressed in the epithelium, and to a lesser degree in the lamina propria, but was absent in goblet cells. In the main olfactory epithelium, virtually all cells in the epithelial layer and select cells associated with Bowman's glands expressed cBD103. We also showed that the ΔG23 mutation did not appreciably alter the antimicrobial activity of the peptide against several species of microorganisms tested in nutrient-rich or minimal media or minimal media with salt added. Moreover, we showed antimicrobial activity in minimal media did not necessarily predict the inhibitory action of the peptide in nutrient-rich media. Both forms of cBD103 caused ultrastructural changes (membrane blebbing, condensation of intracellular contents and cell wall lysis) in Escherichia coli and Staphylococcus aureus. As a ligand of the melanocortin receptors, we showed that cBD103ΔG23 increased ERK1/2 activation and cAMP accumulation when bound to the human or canine melanocortin-4 receptor, acting as a weak allosteric agonist.     

兔中性粒细胞防御素的抗出血热病毒研究

从新西兰白兔的中性粒细胞胞浆颗粒中提取兔防御素，测定其体外抑制流行性出血热病毒（ＥＨＦＶ）的活性实验，结果表明：免防御素对ＥＨＦＶ感染Ｖｅｒｏ细胞无阻断作用，而对ＥＨＦＶ有直接杀灭作用，２０μｇ／ｍｌ时其抗病毒指数为１９６。同时，兔防御素对ＥＨＦＶ在Ｖｅｒｏ中的增殖有抑制作用，４０μｇ／ｍｌ时其抗病毒指数为２０８。     

大鼠β-防御素-2的cDNA克隆及其在气管组织的固有表达

目的　研究大鼠β-防御素基因表达调控分子机制。方法　采用RT -PCR技术在Wistar大鼠气管组织的总RNA中扩增其特异cDNA片段 ,然后进行克隆扩增、限制性内切酶谱分析和DNA序列测定。结果　在大鼠气管组织扩增出长度为 2 1 7bp的cDNA片段 ,其RT -PCR产物经测序分析证明是大鼠β-防御素 - 2cDNA。结论　大鼠β -防御素 - 2基因在气管组织呈固有表达 ,认为与呼吸系统的抗微生物防御机制密切相关。     

昆虫防御素A的分子改造及其在噬菌体表面的展示

目的：实现对昆虫防御素A的分子改造及改造后分子的噬菌体表面展示，为进一步设计构象型肽库，研究分子间相互作用奠定基础。方法：分子改造包括以下内容；分子截短，突变碱性氨基酸，减少半胱氨酸的数目等。利用InsightⅡHomology系统对改造后分子结构进行预测。然后再将合成的改造蛋白基因插入到pCANTAB5E载体中，采用Western印迹鉴定该蛋白在噬菌体表面展示。结果与结论：模建结果显示改造后蛋白仍保持原有三维结构不变，并且成功实现了昆虫防御素分子在噬菌体表面的展示。     

以防御素HD5为效应分子EGFR靶向融合蛋白构建及其抗胰腺癌细胞株活性研究

目的 人α防御素主要由中性粒细胞(HNP1、HNP2和HNP3)和小肠潘氏细胞(HD5和HD6)分泌.HNP1-3可以被作为肿瘤标志物,小剂量的α防御素可以促进肿瘤的增长,大剂量的α防御素可以裂解杀死肿瘤细胞.制备人防御素(human defensin-5,HD5或D5)、力达霉素辅基蛋白(lidamycin apoprotein,LDP)与EGFR配体寡肽(EGFR-di-recting ligand peptide,Ec)组合的融合蛋白Ec-LDP-D5,并初步探讨其抗胰腺癌活性.方法 采用基因工程的方法制备融合蛋白基因,并将其表达纯化得到融合蛋白Ec-LDP-D5.融合蛋白与细胞表面EGFR的亲和活性采用ELISA和细胞免疫荧光化学法;采用CCK-8法测定融合蛋白对人胰腺癌细胞PNAC-1和Aspc-1的体外杀伤活性;流式细胞术检测Ec-LDP-D5蛋白对细胞凋亡的影响.结果 成功构建并表达融合蛋白Ec-LDP-D5,产物主要以包涵体的形式存在,目的蛋白经纯化、复性后,每升发酵液可以获得1 mg纯度达到85％.Ec-LDP-D5可与高表达EGFR的PNAC-1和Aspc-1细胞表面结合.Ec-LDP-D5对PNAC-1和Aspc-1肿瘤细胞有强烈的杀伤作用,Ec-LDP-D5蛋白对Aspc-1肿瘤细胞抑制率为76.8％,与Ec-LDP的抑制率49.4％相比,差异有统计学意义,P=0.038;对PANC-1细胞抑制率为64.6％,与Ec-LDP的抑制率28.3％相比,差异有统计学意义,P=0.042.Ec-LDP-D5在8μmol/L的浓度下即可强烈的诱导细胞发生凋亡,细胞凋亡率(24.6±0.56)％,与Ec-LDP的凋亡率相比P=0.011.结论 本实验制备的防御素融合蛋白Ec-LDP-D5可以与高表达EGFR的PNAC-1和Aspc胰腺癌细胞结合,对胰腺癌细胞具有强烈的杀伤活性和诱导凋亡能力,具有发展为抗肿瘤靶向药物的潜能.     

防御素在牙周组织的分布及功能

防御素是近年来发现的一类带正电荷的小分子多肽，具有抗细菌、抗病毒、抗真菌及抗肿瘤等活性，以抗菌活性尤为突出。牙周病是细菌感染性疾病，菌斑微生物是牙周病的始动因子。本文从防御素在牙周组织中的分布及其功能两方面进行综述，了解防御素在牙周病发生发展中的地位与研究现状。     

IgA肾病患者中人防御素5的临床价值

目的 探讨人防御素5(HD5)与IgA肾病(IgAN)患者临床病理表现及异常糖基化IgA1(Gd-IgA1)之间的关系。方法 选取2021年1月至2021年7月苏州大学附属第一医院原发性IgAN患者50例为IgAN组,选取同期在本院接受健康体检者50例为正常对照组,使用酶联免疫吸附法(ELASE)检测研究对象血清Gd-IgA1和HD5水平,分析IgAN患者血清HD5水平与临床、病理及Gd-IgA1水平的关系。结果 与正常对照组相比,IgAN患者血清HD5及Gd-IgA1水平均升高(P<0.05)。IgAN患者血清HD5水平与Gd-IgA1成正相关,与估算肾小球滤率(eGFR)成负相关。IgAN患者肾小管萎缩/间质纤维化程度越高,血清HD5水平越高(P<0.05)。结论 HD5在IgAN患者中表达增加,并可能参与患者疾病进展。     

Alpha Defensin: A Diagnostic Accuracy Depending on the Infection Definition Used

BackgroundThe purpose of this study was to evaluate the alpha defensin qualitative detection (ADLF) sensitivity and specificity as compared with 3 standard classifications in the diagnostic management of chronic prosthetic joint infections.Materials and methodsA multicenter cohort of 136 patients with a painful arthroplasty was classified into either infected or noninfected according to the Musculoskeletal Infection Society (MSIS) score, Infectious Diseases Society of America (IDSA) score, European Bone and Joint Infection Society (EBJIS) score. The sensitivity and specificity of the ADLF test were calculated for each score. Spearman’s correlations between all scores were then analyzed, and multiple logistic regression was applied to identify independent variables strongly connected to the prosthetic joint infection probability.ResultsThe EBJIS score was positive in 68 patients, IDSA score in 50 patients, MSIS score in 41 patients, and ADLF in 40 patients. The ADLF sensitivity was 87.8% compared with MSIS, 70% compared with IDSA, and 55.8% compared with EBJIS. The ADLF specificity was in the range of 94%-97%. A good correlation was observed between synovial fluid cultures and ADLF (r = 0.73). Low to excellent correlations were recorded between ADLF and the EBJIS (r = 0.58), IDSA (r = 0.68), and MSIS (r = 0.84) scores. The synovial fluid’s white blood cell count was proven to be the biological test that most influenced the probability of a positive culture (P value: .005).DiscussionThe ADLF sensitivity was variable, whereas its specificity was excellent. The EBJIS score results significantly differed from those obtained via cultures, which possibly explains the ADLF low sensitivity compared with that of the EBJIS score.     

Antibacterial peptide defensin is involved in midgut immunity of the soft tick,Ornithodoros moubata

Two defensin genes A and B were previously demonstrated to be up-regulated by blood feeding in the soft tick, Ornithodoros moubata [Nakajima et al. (2001) Two isoforms of a member of the arthropod defensin family from the soft tick, Ornithodoros moubata (Acari: Argasidae). Insect Biochem Mol Biol 31: 747-751]. In this study, two defensin isoforms C and D similar to defensins A and B were newly cloned. A total of four defensins have been identified in O. moubata. All four Ornithodoros defensins are coded as prepro-defensins. Ornithodoros defensin genes consist of four exons and three introns, an organization reported in mussel defensins but not insect defensins. Ornithodoros defensin C and D genes are predominantly expressed in the midgut and up-regulated in response to blood feeding. The mature peptide of the previously cloned Ornithodoros defensin A was purified from the midgut lumen, indicating defensin is secreted into the midgut. These findings confirm the involvement of Ornithodoros defensin in midgut immunity.