穿膜肽-β-葡萄糖苷酶偶联物的制备及活性鉴定

目的 制备穿膜肽-β-葡萄糖苷酶偶联物并鉴定其穿膜活性和酶活性.方法 采用异型双功能交联剂N-琥珀酰亚胺基-3-（2-吡啶二硫）-丙酸酯（SPDP）将穿膜肽·增强型绿色荧光蛋白融合蛋白（CPPs-EGFP）、β-葡萄糖苷酶进行共价交联,经Ni-NAT Superflow Cartridge、Sephadex G-75层析柱分离纯化.采用还原型和非还原型聚丙烯酰胺凝胶电泳（SDS-PAGE）对偶联物进行鉴定；偶联物与膀胱癌EJ细胞共同孵育,置于荧光显微镜下观察,鉴定其穿膜活性；采用β-葡萄糖苷酶测试盒（DBGD-100）鉴定其酶活性.结果 偶联物SDS-PAGE非还原型电泳高分子量端可见明显条带,而还原型电泳分别在穿膜肽和β-葡萄糖苷酶分子量相应位置可见明显条带；在荧光显微镜下与偶联物共同孵育后的膀胱癌EJ细胞内可见明显绿色荧光；用β-葡萄糖苷酶测试盒（DBGD-100）测定,偶联物仍然保持良好的酶活性,约为同等浓度β-葡萄糖苷酶活性的80％.结论 应用SPDP成功制备了穿膜肽-β-葡萄糖苷酶偶联物,并且偶联物保持良好穿膜活性和酶活性.该方法有助于解决酶·前药系统药物难以进入细胞内的问题.     

中东呼吸综合征冠状病毒量子点免疫层析试纸的制备

目的制备一种能快速检测中东呼吸综合征冠状病毒(MERS-CoV)的量子点免疫层析试纸,以期用于MERSCoV感染检测。方法采用EDC偶联法将羧基化CdTe/ZnSe量子点与抗MERS-CoV RBD的单克隆抗体(mAb/MERS-CoV)共价偶联,通过斑点免疫印迹检验偶联物的活性。以CdTe/ZnSe量子点标记的mAb/MERS-CoV作为荧光标记物,采用免疫层析技术制备量子点免疫层析试纸,用于MERS-CoV检测。并对该方法进行特异性、敏感性评价。结果 CdTe/ZnSe量子点可偶联mAb/MERS-CoV,且偶联物具有良好的生物活性。以其作为荧光标记物建立的量子点免疫层析试纸可特异性检测MERS-CoV假病毒,最低检测限为2×53 TCID50/ml,其他对照病毒检测均阴性。结论制备的MERS-CoV量子点免疫层析试纸具有简便、快速、特异等优点,可用于MERS-CoV感染快速诊断和流行病学调查。     

中东呼吸综合征冠状病毒量子点免疫层析试纸的制备北大核心CSCD

目的制备一种能快速检测中东呼吸综合征冠状病毒(MERS-CoV)的量子点免疫层析试纸,以期用于MERSCoV感染检测。方法采用EDC偶联法将羧基化CdTe/ZnSe量子点与抗MERS-CoV RBD的单克隆抗体(mAb/MERS-CoV)共价偶联,通过斑点免疫印迹检验偶联物的活性。以CdTe/ZnSe量子点标记的mAb/MERS-CoV作为荧光标记物,采用免疫层析技术制备量子点免疫层析试纸,用于MERS-CoV检测。并对该方法进行特异性、敏感性评价。结果 CdTe/ZnSe量子点可偶联mAb/MERS-CoV,且偶联物具有良好的生物活性。以其作为荧光标记物建立的量子点免疫层析试纸可特异性检测MERS-CoV假病毒,最低检测限为2×53 TCID50/ml,其他对照病毒检测均阴性。结论制备的MERS-CoV量子点免疫层析试纸具有简便、快速、特异等优点,可用于MERS-CoV感染快速诊断和流行病学调查。     

阿霉素—转铁蛋白受体单抗偶联物对人肝细胞癌导向治疗的实验研究

以ＤｅｘｔｒａｎＴ１０为偶联剂，将阿霉素与抗转铁蛋白受体单抗ＷｕＴ９交联，以此偶联物及对照制剂分别用于荷人肝细胞癌裸鼠的实验治疗。结果表明，无论从潜伏期还是从肿瘤生长速度比较，导向治疗组均优于各对照组。此外，各组荷瘤鼠ＮＫ细胞活性测定表明，导向制剂对宿主免疫机能的抑制作用最小。提示：阿霉素─转铁蛋白受体单抗偶联物可用于人肝细胞癌的生物导向治疗。     

日本血吸虫组织蛋白酶的研究

目的：鉴别日本血吸虫组织蛋白酶类型及其水解蛋白活性。方法：收集日本血吸虫成虫肠道排出物，用含精氨酰键的特异性人工合成底物识别排出物中组织蛋白酶的类型及活性。结果：成虫肠道排出物能降解组织蛋白酶Ｂ的特异性底物７－氨基－４－甲基香豆素（Ｚ－Ａｒｇ－Ａｒｇ－ＡＭＣ）和组织蛋白酶Ｂ及Ｌ的共同底物Ｚ－Ｐｈｅ－Ａｒｇ－ＡＭＣ。组织蛋白酶Ｌ的抑制剂Ｚ－Ｐｈｅ－Ｐｈｅ－ＣＨＮ２能部分抑制排出物对Ｚ－ｐｈｅ－Ａｒｇ－ＡＭＣ的降解作用。日本血吸虫组织蛋白酶Ｂ／Ｌ的最适ｐＨ为５．０－５．５。结论：日本血吸虫成虫排出物具有组织蛋白酶Ｂ和Ｌ样活性。     

用多肽-蛋白偶联方法制备抗恶性疟原虫抗体

目的介绍一种多肽-蛋白偶联的流程,并用多肽-蛋白偶联产物制备抗疟原虫抗体。方法用化学连接剂Sulfo-EMCS在作为载体蛋白的铜绿假单胞菌重组去毒外毒素(rEPA)上加马来酰亚胺基团,用间接Ellman反应测定载体蛋白上所加的马来酰亚胺基团数量。用马来酰亚胺修饰的载体蛋白滴定Pfs48/45-158多肽[含恶性疟原虫表面蛋白48/45(Pfs48/45)第158～173氨基酸序列,其N末端带有一个半胱氨酸残基],绘制滴定曲线并用线性回归进行曲线拟合,根据滴定曲线确定理论滴定终点,计算多肽与载体蛋白的偶联比(每摩尔载体蛋白所能结合的多肽的摩尔数)。用过量的Pfs48/45-158多肽与马来酰亚胺修饰的rEPA进行反应,规模制备Pfs48/45-158-rEPA多肽-蛋白偶联物,偶联物用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)进行鉴定。用所制备的Pfs48/45-158-rEPA偶联物免疫BALA/c小鼠,制备免疫血清。用ELISA测定免疫小鼠血清抗Pfs48/45-158多肽的抗体效价,用免疫荧光试验(IFA)测定免疫血清识别疟原虫的能力。结果通过化学连接剂在每摩尔rEPA上添加约6.94摩尔的马来酰亚胺基团。制备了偶联比约为7.33的Pfs48/45-158-rEPA多肽-蛋白偶联物。偶联物免疫小鼠激发出抗Pfs48/45-158多肽的高抗体应答,免疫血清抗Pfs48/45-158多肽的效价为12 500 ELISA单位(即吸光度A405值为1时的血清稀释度倒数),同时免疫血清可识别疟原虫。结论多肽-蛋白偶联是一种可用于制备抗疟原虫抗体的便捷方法,间接Ellman检测、滴定反应和SDS-PAGE分析构成了多肽-蛋白偶联物制备的质控方法,可更好地保证多肽-蛋白偶联物的质量和稳定性。     

家蝇消化腺抗菌肽生物活性的研究

目的研究家蝇消化腺抗菌肽抗菌活性和细胞毒性。方法针刺细菌感染法诱导家蝇幼虫表达抗菌肽,采用显微解剖的方法解剖并收集成蝇中肠,用超声破壁低温离心等技术提取、纯化抗菌肽,用提取的抗菌肽进行抑菌试验及细胞毒性试验。结果金黄葡萄球菌诱导的家蝇中肠抗菌肽抑菌环直径为1.3 cm,正常成蝇中肠抗菌肽抑菌环直径为1.1 cm,青霉素及蛆血淋巴抗菌肽抑菌环直径分别为1.0和<0.5 cm。细胞毒性试验中,各浓度抗菌肽组细胞生长正常。讨论中肠抗菌肽抑菌活性优于蝇蛆血淋巴抗菌肽的抑菌活性,且细胞毒性低。     

帕尼培南/倍他米隆治疗老年人感染性疾病

帕尼培南／倍他米隆(panipenem／betamipron)是日本三共株式会社研究新开发的一种碳青霉烯类抗生素，本品用panipenem(cs-533，PAPM)与betamipron(cs-433，BP)1：1配伍而成，具有广谱杀菌作用，抗菌活性甚强，对需氧革兰阴性和阳性菌以及厌氧菌均具良好抗菌作用，在血药浓度刚高于细菌的平均抑菌浓度(MIC)时能迅速杀菌，在最初的3h接触中，可杀灭99％以上细菌，抗菌活性可达     

尾加压素Ⅱ及其与动脉粥样硬化关系的研究进展

尾加压素Ⅱ（UⅡ）最早是从鱼的脊髓尾部下垂体中分离出的生长抑素样环肽,是目前已知最强的缩血管活性肽。人体中一种孤立的受体G蛋白偶联受体14（GPR14）是UⅡ的特异性受体,主要分布于心血管系统。     

枸骨叶不同溶媒萃取物对二种念珠菌作用的研究

据本草纲目记载,枸骨叶味苦,微涩,性寒。有清热解毒、止渴生津的功能。我们证实,枸骨叶的化学萃取物对金黄色葡萄球菌、沙门氏菌、大肠杆菌等细菌均有一定的抑菌活性。本实验旨在进一步探讨枸骨叶不同萃取物成分对病原性念珠菌的生长有无抗菌活性,以期能开发对临床有实用价值的天然抗真菌药物。     

The immunoneuroendocrine role of melatonin

Abstract: A tight, physiological link between the pineal gland and the immune system is emerging from a series of experimental studies. This link might reflect the evolutionary connection between self-recognition and reproduction. Pinealectomy or other experimental methods which inhibit melatonin synthesis and secretion induce a state of immunodepression which is counteracted by melatonin. In general, melatonin seems to have an immunoenhancing effect that is particularly apparent in immunodepressive states. The negative effect of acute stress or immunosuppressive pharmacological treatments on various immune parameters are counteracted by melatonin. It seems important to note that one of the main targets of melatonin is the thymus, i.e., the central organ of the immune system. The clinical use of melatonin as an immunotherapeutic agent seems promising in primary and secondary immunodeficiencies as well as in cancer immunotherapy. The immunoenhancing action of melatonin seems to be mediated by T-helper cell-derived opioid peptides as well as by lymphokines and, perhaps, by pituitary hormones. Melatonin-induced-immuno-opioids (MHO) and lymphokines imply the presence of specific binding sites or melatonin receptors on cells of the immune system. On the other hand, lymphokines such as -γ-interferon and interleukin-2 as well as thymic hormones can modulate the synthesis of melatonin in the pineal gland. The pineal gland might thus be viewed as the crux of a sophisticated immunoneuroendocrine network which functions as an unconscious, diffuse sensory organ.     

Changes in connexin43, the gap junction protein of astrocytes, during development of the rat pineal gland

Abstract: The abundance of gap junctions between rat pineal astrocytes formed by connexin43 (Cx43) was studied during development. Levels and distribution of Cx43 were measured by immunoblotting and indirect immunofluorescence, respectively. The amount of Cx43 in cells located within the gland was low until about the 7th postnatal day and increased to adult values between the 14th and 21st days postpartum. Although astrocytes, recognized by their vimentin immunoreactivity, were scarce before birth, they were abundant by the 7th postnatal day suggesting that the low levels of Cx43 found at this age corresponded to a low expression of this protein. Localization of the immunoreactivity to Cx43 and vimentin showed a close correlation, indicating that mature or immature pineal astrocytes form gap junctions made of Cx43. Since Cx43 levels attained their adult values at about the time the innervation and the functional state of the gland reached maturity (2–3 weeks after birth), it is proposed that astrocyte gap junctions are involved in the function of the adult rat pineal gland.     

Response of rat pineal melatonin to calcium, magnesium, and lithium is circadian stage dependent

Abstract: Herein we documented the response of pineal melatonin production to electrolytes known to be effective on pineal function in view of a possible circadian stage dependence. We studied the release of melatonin by perifused rat pineal glands at 2 different circadian stages corresponding to the middle of the light and dark periods, i.e., respectively, 7 and 19 HALO (Hours After Light Onset, L:D = 12:12). The initial efflux rates were, as expected, much higher in the perifusates of glands removed from rats sacrificed during the dark phase than of those removed during the light phase. After 3 hr of perifusion, melatonin release reached similar levels which were found constant up to the 8th hr of perifusion, whatever the circadian stage. Perifusion of the glands with physiological concentrations for the rat of calcium (5.2 mmol/1) and magnesium (1.34 mmol/1) resulted in a stimulatory effect on the pineal glands removed from rats sacrificed in the middle of the dark period (19 HALO), whereas no effects were observed on the pineal glands removed from rats sacrificed during the light (7 HALO). Lithium (0.28 and 0.55 mmol/1) was ineffective on melatonin release in pineal glands removed 7 and 19 HALO. Our results show differences in the initial efflux rates of melatonin and in the response of perifused pineal glands to calcium and magnesium according to the circadian stage.     

Conservative management of perforated duodenal diverticulum： A case report and review of the literature

Duodenal diverticula are a relatively common condition. They are asymptomatic, unless they become complicated, with perforation being the rarest but most severe complication. Surgical treatment is the most frequently performed approach. We report the case of a patient with a perforated duodenal diverticulum, which was diagnosed early and treated conservatively with antibiotics and percutaneous drainage of secondary retroperitoneal abscesses. We suggest this method could be an acceptable option for the management of similar cases, provided that the patient is in good general condition and without septic signs.     

Presence of immunoreactive growth hormone and prolactin in the ovine pineal gland

Abstract: The use of antisera raised against bovine growth hormone (GH) and ovine prolactin (PRL) enabled the detection of related immunoreactive (ir) sequences of proteins in ovine pineal tissue. The isolation of PRL-like ir-material was accomplished using a 0.25 M ammonium sulphate (pH 5.5) extraction followed by ethanol precipitation, whereas the resulting 2.0 M ammonium sulphate (pH 7.0) precipitate contained a GH-like immunoreactivity. Gel chromatography of the GH-like immunoreactivity (Sephadex G-100) indicated the presence of several GH-like fragments ranging in the M_r range of 7,000 to 55,000. Analyses of the PRL-like ir-material found in pineal tissue on HPLC using a TSK 545-DEAE column led to the resolution into a single peak of immunoreactivity. A single peak of activity was also observed following chromatofocusing and hydrophobic interaction chromatography of the ir-peak from the TSK 545-DEAE column. The PRL-like ir-material inhibited the binding of [¹²⁵I]ovine PRL-S14 to anti-ovine PRL antibodies without showing an affinity for binding to anti-rat PRL or anti-bovine GH antibodies. Scatchard analysis of the binding of pineal PRL-like ir-material and pituitary ovine PRL-S14 to liver membranes from day-20 pregnant rats revealed similar affinity constants (K_a of 4.7 ± 0.2 × 10⁹ M^-1). In addition, the replication of Nb 2 Node rat lymphoma cells was stimulated by pineal PRL-like ir-material, an effect known to be specific for lactogenic hormones. The pineal PRL-like immunoreactivity appeared on sodium dodecyl sulfate polyacrylamide gels as a single major band of M_r 24,000. The functional status of PRL-and GH-like ir-material in the ovine pineal remains to be determined, but evidence is presented that the overall protein synthesis rate of the rat pineal responded to circulating concentrations of PRL.     

Microbiology of human immunodeficiency virus anorectal disease

PURPOSE: Individuals who are seropositive for the human immunodeficiency virus are at high risk for opportunistic infection and anorectal disorders. Little prospective information is available regarding anorectal pathogens in these patients. METHODS: One hundred sixty-three HIV-seropositive patients presented to the colorectal clinic between 1989 and 1992. Forty-seven (29 percent) patients were thought to have an infectious process and were prospectively studied using a standardized multiculture protocol. RESULTS: Mean age was 33 (range, 19–59) years. All were male; high-risk behavior accounted for 87 percent of HIV transmissions. Presenting complaints included anorectal pain (79 percent), pus per anum (28 percent), and blood per anum (26 percent). Examination revealed perianal tenderness (60 percent), condyloma (38 percent), perianal ulcers (38 percent), and anal fissures (34 percent). Sixty-six sets of cultures were performed; 28 patients had one set, 15 had two sets, and 4 had three sets. Thirty-two of these 47 patients (68 percent) had positive cultures including herpes (50 percent), cytomegalovirus (25 percent),Neisseria gonorrhoeae (16 percent), chlamydia (16 percent), acidfast bacilli (2 percent), and others (9 percent). Six of 32 patients with positive cultures had more than one organism cultured. Sixteen (50 percent) patients with positive cultures were treated medically, 8 (25 percent) were treated surgically and 8 (25 percent) were treated with both modalities. Sixty-one procedures were performed on 17 patients for condylomata. Eighteen patients had 20 procedures for abscesses, 50 percent of whom had positive cultures for other than common bowel flora; all improved. Fourteen patients underwent 33 procedures for perianal fistulas.Mycobacterium fortuitum was cultured from one patient who required 13 procedures for abscesses and fistulas. Forty-five (96 percent) patients were followed for an average of 12.5 months ±2.9 SEM (range, 1–94 months). Symptoms were improved or resolved in 22 of 32 (69 percent) patients with positive cultures and in 11 of 13 (84 percent) with negative cultures. CONCLUSIONS: Specific pathogens may often be identified in human immunodeficiency virus-seropositive patients with anorectal disorders if aggressively sought. Although patients without specific pathogens identified may be expected to improve with planned empiric treatment, positive identification allows more directed therapy.