中国鼠疫菌随机引物扩增多态性指纹图谱

目的 对中国不同鼠疫自然疫源地鼠疫菌染色体的分析和比较发现其近缘关系进而从分子水平进行分型。方法 用随机扩增多态性DNA(RAPD)技术。结果 第1条引物将中国的鼠疫菌分为11个类型，每个类型都有一定的条带规律和地理分布范围。第2条引物将我国的鼠疫菌分为5个类型。结论 随机扩增多态性DNA(RAPD)可以从分子水平用于我国鼠疫菌的分型。     

肺炎支原体P1蛋白结构基因片段的制备及C末端基因片段克隆的研究

目的 制备肺炎支原体P1蛋白结构基因,并在大肠杆菌中克隆肺炎支原体P1蛋白C末端基因片段,为P1蛋白基因片段的扩增、表达及探讨C末端基因片段功能打基础。方法 PCR扩增方法获取P1结构基因。扩增产物用P1蛋白基因特异引物扩增鉴定。用Sal Ⅰ和EcoR Ⅰ双酶酶切消化方法制备P1蛋白C末端基因片段,并与pUC19DNA连接,转入大肠杆菌JM109菌株。用X－gal平板及质粒图谱分析方法筛选重组克隆株,再用限制性核酸内切酶酶切图谱分析鉴定。结果 经PCR扩增MP DNA获得一条5．0kbDNA片段,用P1基因区特异引物扩增鉴定获得阳性结果。重组质粒限制性内切酶批纹图谱显示出两条带,一条为pUC19载体DNA带,另一条是1kb的输入片段。结论 获得肺炎原体P1蛋白结构基因及含P1蛋白C末端DNA片段的重组克隆株。     

用4对引物聚合酶链式反应检测鼠疫耶尔森氏菌的实验研究

目的用4对引物聚合酶链式反应(以下简称4-Prim er-PCR)来检验鼠疫耶尔森氏菌,对于探索鼠疫的快速诊断方法将起到推动作用。方法选择不同疫源地的40株鼠疫耶尔森氏菌;目的基因选择于与鼠疫耶尔森氏菌毒力因子有关的FI抗原质粒基因cafI鼠疫杆菌素Ⅰ基因、与色素沉着有关的质粒基因hm s以及染色体基因inv。结果该法所有的鼠疫耶尔森氏菌全部出现4对引物的预期扩增带;该试验在4 h内完成;结论四对引物鼠疫PCR法具有快速、特异、敏感等优点,可以用于鼠疫的快速诊断和流行病学调查。     

鼠疫耶尔森氏菌16S-23SrRNA基因间区分析

目的　发展一种快速准确的鉴定鼠疫菌的方法。方法　 Ｐ Ｃ Ｒ 反应加限制性内切酶消化的方法。结果　对来自不同疫源地的 １０３ 株鼠疫菌 １６ Ｓ２３ Ｓｒ Ｒ Ｎ Ａ 基因间区进行扩增,均可扩出两条长为 １ １４６ｂｐ 及 １ ０９０ｂｐ 的片段,扩增产物用限制性内切酶 Ｔａｑ Ｉ, Ｍ ｓｐ Ｉ消化后的酶切图谱相同。而对照菌株小肠结肠炎耶尔森氏菌,鼠伤寒沙门氏菌,大肠杆菌,痢疾杆菌,霍乱弧菌的扩增产物及酶切图谱与鼠疫菌均不相同。结论　鼠疫菌 １６ Ｓ２３ Ｓｒ Ｒ Ｎ Ａ 基因间区高度同源,基本为两种类型,长度分别为 １ １４６ｂｐ 及 １ ０９０ｂｐ,其他菌株与其明显不同;该方法将有助于快速准确的鉴定鼠疫菌。     

PCR-RFLP技术用于鉴别赫坎按蚊复合体近缘种按蚊的研究

目的区别赫坎按蚊种团内近缘种.方法应用聚合酶链式反应连接的限制性片段长度多态性方法(PCR-RFLP)技术,对辽宁省现场捕获的按蚊用特异性ITS2引物进行PCR扩增,限制性内切酶RsaⅠ和HinfⅠ消化,琼脂糖凝胶电泳分析.结果中华按蚊的PCR扩增产物能被限制性内切酶RsaⅠ酶切成350 bp和200 bp两条酶切DNA条带;嗜人按蚊核糖体DNA(rDNA)的PCR扩增产物能被限制性内切酶HinfⅠ酶切成410 bp的酶切DNA条带; 雷氏按蚊的ITS2基因PCR扩增产物能分别被限制性内切酶RsaⅠ和HinfⅠ酶切,分别显示350 bp和400 bp的酶切DNA条带;八代按蚊的PCR扩增产物没有显示明显的限制性内切酶RsaⅠ或HinfⅠ酶切条带.结论依据rDNA的ITS2区段基因特征建立的PCR-RFLP技术可用于鉴别赫坎按蚊种团的中华按蚊、嗜人按蚊、雷氏按蚊和八代按蚊4个近缘种按蚊.     

鼠疫耶尔森菌基因组中IS100插入位点的特异性分析

目的 通过鼠疫耶尔森菌基因组中插入序列IS100位点的特异性研究,对我国鼠疫菌基因组的构成类型和特点进行分析.方法 以鼠疫菌东方变种CO92全长序列为参考,选择5个位置的IS100位点,在IS100基因相邻两外侧设计1对引物,在IS100基冈内设汁1对向外引物使其分别能够与两外侧引物扩增.对来自13个生态型的91株鼠疫菌和1株假结核耶尔森菌进行PCR扩增,产物进行克隆测序分析,同时把各菌株的IS100位点情况标记在我国鼠疫疫源地类型慨图上二进行分析.结果 实验所用91株鼠疫菌在所选定IS100位点出现含有和不含IS100的插入序列,基因片段组成相对CO92序列已发生改变,而且这些IS100基因型的组成在我国鼠疫自然疫源地中呈现区域性分布.结论 IS100基因型别分布与我国鼠疫菌疫源地分型分布基本一致,并直接反映了鼠疫菌基因组某特定位点的基因组成状况及其在进化过程中的高度流动性.     

弓形虫P30抗原基因的PCR扩增及克隆的初步研究

根据已发表的弓形虫主要表面抗原Ｐ３０基因序列，自行设计合成了一对引物，分别在其正义链和反义链的５′末端加上了ＢａｍＨⅠ和ＥｃｏＲⅠ的酶切位点，利用ＰＣＲ技术获取Ｐ３０抗原基因整个开放阅读框架的碱基序列。ＰＣＲ产物经ＢａｍＨⅠ和ＥｃｏＲⅠ双酶切后，与经同样两种内切酶切割的质粒载体ｐＢｌｕｅｓｃｒｉｐｔＳＫ连接构成重组质粒ｐＢｌｕｅｓｃｒｉｐｔＳＫ－Ｐ３０，转化大肠杆菌ＤＨ５α。通过遗传标记筛选、ＰＣＲ扩增及酶切分析对重组子进行了鉴定，为进一步选择真核系统或原核系统体外表达该抗原打下了必要的基础。     

不同PCR联合用于鼠疫耶尔森菌的检测

目的 建立一种用4对不同引物快速鉴定鼠疫耶尔森菌的PCR方法。方法 分别采用4对针对V抗原，cafl，pla和inv4种基因的引物，在4只管中进行相同循环参数的扩增。结果 用这4对引物能特异性地扩增出鼠疫耶尔森菌217，478，524，295bp的目的DNA片段。结论 该法可用于鼠疫耶尔森菌简便、快速和准确的检测。     

乙型肝炎病毒全基因组研究的新方法

目的为从全基因水平研究乙型肝炎病毒变异与致病性的关系，建立血清标本经聚合酶链反应（ＰＣＲ）加酶切方法扩增及克隆ＨＢＶ全基因组的新方法。方法设计了含ＳｐｅⅠ，ＳａｌⅠ及ＳａｐⅠ酶切点的引物，分别扩增１．１５ｋｂ和２．０５ｋｂ的单链及双链ＤＮＡ区。经酶切及连接后获得ＨＢＶ全基因克隆。结果用新建立的方法直接从少量血清中克隆了ＨＢＶ全基因，转染ＨｅｐＧ２细胞可表达抗原并有胞内复制。结论该法可用于对临床上ＨＢＶ毒株的基因结构和功能的研究     

应用PCR/RFLP对山东地区恙虫病东方体的基因型分析

目的　鉴定山东地区恙虫病东方体的基因型。方法　采用聚合酶链反应 /限制性片段长度多态性分析 (PCR/RFLP)技术对恙虫病东方体山东分离株、小盾纤恙螨匀浆及恙虫病病人全血中提取的DNASta5 6基因进行分析研究 ,并与NestedPCR基因分型的结果进行比较。结果　山东地区 35份标本 ( 2 3株恙虫病东方体山东分离株、2份采自黑线姬鼠体外的小盾纤恙螨匀浆及 10份恙虫病人全血 )提取的DNA群引物扩增产物经HinfⅠ、HhaⅠ、SnaBⅠ酶切后呈现 2种不同的RFLP图谱 ,经与已知序列的Ot内切酶图谱相比 :33份Ot内切酶图谱与文献报告的日本地方株—Kawasaki株具有相似的酶切图谱 ,但缺乏HhaⅠ的酶切位点 ;另 2份与Karp参考株具有相同的酶切图谱。该结果与NestedPCR基因分型的结果相一致。结论　山东地区恙虫病东方体流行株主要基因型与日本地方株Kawasaki型类似 ,但与日本地方株存在遗传差异 ;同时还有Karp型Ot存在。采用PCR/RFLP方法可以准确对Ot-Sta5 6基因分型。     

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.