日本血吸虫酚氧化酶的组化定位研究

目的观察酚氧化酶(phenol oxidase,PO)在日本血吸虫成虫的组织学分布.方法酚氧化酶组化方法:将42 d虫龄的日本血吸虫活成虫置含25 mmol/L邻苯二酚的磷酸盐缓冲液(pH 6.8)中,37℃孵育30 min后,转移虫体至载玻片上,吸去虫体表面液体,将其中一部分虫体置Olym-pus显微镜下观察酚氧化酶在虫体的组织学分布.荧光组织化学方法:经上述过程处理后,在另一部分虫体上滴加2滴含0.05%戊巴比妥钠的PBS溶液,然后置Leica荧光显微镜下观察酚氧化酶在虫体的组织学分布.结果酶组织化学方法显示,日本血吸虫酚氧化酶仅分布于雌虫卵黄腺及子宫内虫卵卵壳表面,呈现棕褐色显色反应;雌虫卵巢和雄虫均未发现酚氧化酶活性.然而,荧光组织化学方法显示,酚氧化酶除主要分布于雌虫卵黄腺及子宫内虫卵卵壳表面,呈现强荧光外,还少量分布于雄虫体壁表层,呈现弱荧光反应.结论不仅日本血吸虫雌虫含有酚氧化酶活性,而且雄虫也含酚氧化酶活性,只不过其含量少、活性低.荧光组织化学方法能更灵敏地显示日本血吸虫酚氧化酶活性,更适用于日本血吸虫酚氧化酶的组织学定位.     

丙烯硫脲对日本血吸虫酚酶抑制作用的组织化学研究

目的 通过组织化学方法研究丙烯硫脲对小鼠体内、体外日本血吸虫成虫酚酶(phenol oxidase，PO)活性的抑制作用。方法 将42d日本血吸虫活成虫置于含25mmol／L邻苯二酚的磷酸盐缓冲液(pH6．8)中，28℃下孵育30min，取出虫体置载玻片上，加2滴含0．1％戊巴比妥钠的PBS溶液，置Leica荧光显微镜下，分别在普通光照和紫外光激发下观察酚酶在虫体内的组织学定位。体外抑制实验时，加底物前先将成虫置于含5mmol／L丙烯硫脲的PBS溶液中，28℃下孵育10min；体内抑制实验时，在小鼠感染日本血吸虫尾蚴20d后，按300mg／kg隔日1次腹腔注射丙烯硫脲。结果 体外抑制实验组雌虫卵黄腺及子宫内虫卵卵壳和雄虫体壁表层的桔黄色荧光或赭褐色颜色反应均基本消失；体内抑制实验组雌虫子宫内未见虫卵或虫卵样物质，仅见大量泥沙样卵黄颗粒，其荧光或颜色反应也基本消失。结论 通过一种更灵敏的显示酚酶活性的组织学定位方法，观察到不仅雌虫含有酚酶，雄虫也有酚酶活性。证明丙烯硫脲对血吸虫成虫的酚酶活性有明显的抑制作用，从而可使小鼠体内血吸虫雌虫虫卵的形成被完全抑制。     

酚酶活性抑制对小鼠体内日本血吸虫产卵的影响

目的观察小鼠体内日本血吸虫雌虫酚酶活性被抑制后对其产卵的影响.方法小鼠感染日本血吸虫尾蚴20d后,按500mg/(kg*d)腹腔注射酚酶抑制剂-丙烯基硫脲,感染后第45d剖杀小鼠,观察感染小鼠体内日本血吸虫雌虫子宫及小鼠肝脏中虫卵的数量及形态结构.结果与感染对照组相比,实验组小鼠雌虫子宫内虫卵数及每克肝组织虫卵数均明显减少.虫卵形态表现异常,主要是缺失硬化的卵壳结构.结论日本血吸虫雌虫酚酶活性被完全抑制后,雌虫不能产卵;不完全抑制则只能产生无正常卵壳的异常虫卵,证明具有活性的酚酶是虫卵正常形成过程中必不可少的成分,提示血吸虫酚酶有可能作为抗血吸虫病的一个新靶位.     

日本血吸虫酚氧化酶天然蛋白分子特征的初步分析

目的初步分析日本血吸虫酚氧化酶(pheno1 oxidase,PO)天然蛋白分子的分子量及其亚基组成.方法将42 d龄活成虫置于含0.05%戊巴比妥钠的RPMI 1640培养基中23 C孵育8 h后,PBS(pH 6.8)洗净,分离雌、雄成虫,研磨呈匀浆,超声粉碎、低温高速离心,取上清(含PO)进行聚丙烯酰胺凝胶电泳(PAGE),分别在两侧切取胶宽约1 cm进行酶染色,然后用解剖刀准确切下相应的未染色的凝胶.经真空冷冻干燥机冻干后碾成粉末,高速电动匀浆成浆糊状,冷浸过夜,高速离心得上清即为日本血吸虫酚氧化酶粗蛋白,进行SDS-PAGE.利用SDS-PAGE与PAGE(点加1孔预染Mark蛋白)的结果,对照分析酚氧化酶蛋白的可能分子量及其亚基组成.结果 PAGE:日本血吸虫雌虫、雄虫酚氧化酶蛋白均表现为迁移率相同的1条主带,其对应分子量大约为81 kD.SDSPAGE:酚氧化酶粗蛋白共出现5条带,分别为81、64、54、41 kD和27 kD.结论初步分析日本血吸虫雌、雄成虫酚氧化酶天然蛋白分子的分子量及其亚基组成基本相同,可能为单体酶(分子量81kD)或由3个27 kD相同亚基构成的同聚体.其确切的分子特征有待于进一步研究.     

酚酶抑制剂对日本血吸虫感染小鼠抗再感染的影响

目的观察雌虫酚酶活性被抑制后,日本血吸虫成虫诱导感染小鼠的伴随免疫作用.方法每只小鼠初次感染日本血吸虫尾蚴(40±1)条,42 d后攻击感染(40±1)条尾蚴,其间初次感染21 d开始,按300 mg/kg的剂量隔日1次腹腔注射丙烯基硫脲,至攻击感染后20 d剖杀小鼠.同时设立实验对照组(未注射丙烯基硫脲组),初次感染对照组及攻击感染对照组.计算各组减虫率并进行统计学处理.结果实验组和对照组减虫率分别为86.4%和36.8%.经χ2检验差异有非常显著意义(χ2=10.7,P<0.01).两组检获虫体总数分别为28.9±10.2和34.8±14.2.经t检验差异也有显著意义(t=1.785,P<0.05).结论日本血吸虫酚酶活性被抑制后,虫卵卵壳形成被抑制,雌虫不能产出正常虫卵或虫卵不能发育成熟.依此建立的新模型提示,无可溶性虫卵抗原(SEA)存在,成虫能诱导小鼠更强的抗再感染作用.     

日本血吸虫酚氧化酶诱导免疫对小鼠保护性的初步研究

目的观察日本血吸虫酚氧化酶的抗原性和诱导宿主抗血吸虫病的免疫保护性作用。方法将含酚氧化酶的上清液进行聚丙烯酰胺凝胶电泳(PAGE)后,分别在两侧切取胶宽约1cm进行酶染色,然后准确切下相应的未染色的凝胶。经真空冷冻干燥机冻干后碾成粉末,高速电动匀浆成浆糊状,冷浸过夜。高速离心得上清即为日本血吸虫酚氧化酶粗抗原。设立3组进行保护性免疫实验A组(实验组)、B组(佐剂对照组)和C组(空白对照组)。初次免疫后第2、4周,各重复1次加强免疫,最后1次免疫后10d按常规收集各组免疫血清。常规ELISA法测定免疫血清效价。然后用新鲜逸出尾蚴(40±1)条/鼠进行攻击感染。6周后,收集成虫计数,常规计算减虫率并检测肝脏虫卵负荷。结果ELISA法测定的酚氧化酶免疫小鼠血清效价在1∶1200以上。日本血吸虫酚氧化酶粗抗原免疫小鼠,其雌虫、雄虫和总虫数的减虫率分别为53.27%、26.76%和39.53%,与C组相比,差异均有极显著意义(P<0.01)。A组减卵率为50.75%,与C组相比,差异有极显著意义(P<0.01)。结论日本血吸虫酚氧化酶具有抗原性,能诱导宿主抗血吸虫感染免疫保护作用。其诱导保护性免疫的机制尚待进一步研究。     

日本血吸虫各期虫体的酶联免疫电转移印迹分析及抗原定位研究

本文采用酶联免疫电转移印迹技术（EITB）分析、比较日本血吸虫不同发育时期虫体特定的组分蛋白分子、雌虫、雄虫和虫卵抗原分别与相应的雌、雄虫和虫卵免疫血清反应呈现17、20和8条蛋白带,这三种抗原与其它不同时期虫体免疫血清反应,三者间及彼此间均出现交叉反应,而雌、雄虫和虫卵抗原与其它寄生虫感染血清作用没发现有叉及反应。应用间接荧光抗体试验（IFA）和免疫酶染色试验（IES）对日本血吸虫抗原进行定位研究,其结果相似。血吸虫主要抗原物质来源于成虫表皮、肠上皮和卵内的毛蚴.     

日本血吸虫酚酶的研究

为了解日本血吸虫成虫的酚酶是否以同工酶的形式存在,利用等电聚焦电泳技术,分离日本血吸虫雌、雄虫及雌雄合抱虫的酚酶同工酶。发现有一组酚酶同工酶,对Dopa、邻苯二酚及对甲酚等底物均呈阳性反应,其中以Dopa作废物时所出现的呈色反应最明显。     

中国与日本不同地域品系日本血吸虫蛋白组分的SDS-PAGE和EITB分析

目的： 补充观察我国浙江、江西、台湾省和日本山梨的日本血吸虫成虫的蛋白组分, 以及上述４ 地和安徽、湖北、四川、云南省血吸虫与广西省钉螺和日本钉螺抗血清的反应性。方法： 采用ＳＤＳ－聚丙烯酰胺凝胶电泳 （ＳＤＳ－ＰＡＧＥ） 和酶联免疫电转移印迹 （ＥＩＴＢ） 分析。结果与结论： 浙江、江西、台湾省和日本的血吸虫隔离群雄虫及雌虫抗原经ＳＤＳ－ＰＡＧＥ后, 以考马斯亮蓝染色结果分别可见７～１７ 条（江西省和日本的血吸虫雄虫条带显色很淡） 及１～６ 条显色带;银染结果分别可见１４～２３条及４～１９ 条（台湾省的血吸虫雌虫仅见１条极淡的）显色带。台湾省血吸虫雄虫与浙江省血吸虫雄虫谱型相似, 但在８１ ｋＤａ以上组分有所不同, 江西省血吸虫雄虫５４ｋＤａ 条带明显, 日本的血吸虫雄虫不仅条带少, 谱型也与浙江、江西及台湾省的血吸虫各异。ＥＩＴＢ结果： 所试血吸虫成虫与广西省钉螺和日本的钉螺抗血清均产生反应, 证明存在交叉反应抗原组分。所试血吸虫成虫与广西省钉螺和日本的钉螺间存在共同抗原组分。     

篙甲醚对日本血吸虫糖代谢酶的影响’

目的 观察篙甲醚(Art)对日本血吸虫糖代谢酶活性的影响.[方法〕小鼠感染日本血吸虫尾坳4-5wk后;1 次灌服Art3 00mg/kg,于治疗后24h ,48h 或72h 剖杀收集雌虫和雄虫,制备虫匀浆上清液,然后进行平板淀粉凝胶电泳,以对9种糖代谢酶活性作定性观察。〔结果〕经Art作用24^48h 后,雌虫的己糖激酶、苹果酸脱氢酶、苹果酸酶和磷酸葡萄糖异构酶活性均有所下降;72h 后,上述4种酶以及醛缩酶、6-磷酸葡萄糖脱氢酶、6-磷酸葡糖酸脱氢酶和6-磷酸甘露糖异构酶的活性均明显降低,甚至完全被抑制。在上述观察时间内,雄虫的醛缩酶、6-磷酸甘露糖异构酶和6-磷酸葡萄塘脱氢酶活性受一定的影响。【结论」Art对日本血吸虫雌虫的糖代谢具有广泛的影响     

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.     

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.     

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.     

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.