墨西哥利什曼原虫无鞭毛体蛋白的基因克隆化与序列分析

目的 克隆墨西哥利什曼原虫（Ｌ．ｍｅｘ）ＷＲ９７２株的无鞭毛体蛋白（ａｍａｓｔｉｎ）的编码基因,并对其同源核苷酸序列进行分析。方法 根据已克隆的亚马逊利什曼原虫无鞭毛体蛋白的编码基因序列,设计并合成成无鞭毛体蛋白基因特异性引物,以墨西哥利什曼原虫ＷＲ９７２株的基因组ＤＮＡ作为模板,进行多聚酶链反应（ＰＣＲ）扩增。将扩增的ＤＮＡ片段克隆到ｐＣＲ２．１Ｔ载体中,进行测序,并对同源的核苷酸序列分析、比较     

亚马逊利什曼原虫无鞭毛体蛋白基因的克隆化与序列分析

目的：克隆亚马逊利什曼原虫（L.ama)无鞭毛蛋白（amastin)的编码基因,并对其同源基因序列进行分析,方法：根据我们首次克隆的硕大利什曼原虫（L.major)无鞭毛体蛋白的编码基因,设计并合成核苷酸序列特异性引物,以亚马逊利什曼原虫基因组DNA为模板,以多聚酶链反应PCR技术扩增无鞭毛体的编码基因DNA片段,并进行核苷酸 列测定以及核苷酸序列的同源性分析。结果：克隆了亚马逊利什曼原虫无鞭毛体蛋白的编码基因,含有单一开放读框,长度为552bp,编码的无鞭毛体蛋白由183个氨基酸残基（aa)组成,亚马逊利什曼原虫与硕大利什曼原虫无鞭毛体蛋白编码基因之间高度同源,在核苷酸与氨基酸残基序列水平上的同源性分别为96％和94％,结论：首次实现亚马逊利什曼原虫无鞭毛体蛋白基因的克隆化。     

利什曼原虫无鞭毛体蛋白的基因克隆化与序列分析

目的 克隆4株利什曼原虫表面无鞭毛体蛋白（amastin）的编码基因,并进行序列分析。方法 根据锥虫（T.cruzi)与利什曼原虫亲缘关系相近的原则,首先以锥虫无鞭毛体蛋白的基因为参考。对GenBank中的dbFST数据库检索,获得硕大利什曼原虫（L.major)一段309核苷酸片段,根据其序列合成探针,对硕大利什曼原虫基因组DNA文库筛选,首先获得硕大利什曼原虫无鞭毛体蛋白编码基因,再以硕大利什曼原虫无鞭 毛体蛋白编码 基因序列为依据,合成特异性引物,以多聚酶链反应（PCR）扩增获得亚马逊利会曼原虫（L.ama.)、巴西利什曼原虫（L.bra.)和墨西哥利什曼原虫（L.mex.)的无鞭毛体蛋白基因。结果 克隆了4株利什曼原虫无鞭毛体蛋白编码的基因。均为国际上首次克隆化基因,已被美国GenBank收录。结论 实现了4株利什曼原虫无鞭毛体 蛋白编码基因的克隆化。     

硕大利什曼原虫无鞭毛体蛋白的基因克隆与序列分析

[目的 ]克隆硕大利什曼原虫无鞭毛体蛋白 (amastin)的编码基因序列。 [方法 ]应用核苷酸序列数据库 (GenBank)和表达序列末端片段数据库 (dbEST)的计算机检索与DNA文库的杂交筛选方法。 [结果 ]从dbEST数据库中获得一段 30 9nt的来源于硕大利什曼原虫的基因片段 ,据此设计探针 ,筛选硕大利什曼原虫的DNA文库 ,获得硕大利什曼原虫无鞭毛体蛋白的编码基因。其开放读码框架由 5 5 2个核苷酸组成 ,编码产物由183个氨基酸残基组成。序列分析表明 ,硕大利什曼原虫与锥虫无鞭毛体蛋白一级结构的同源性为 2 3 5 %。 [结论 ]克隆的基因系硕大利什曼原虫表面蛋白编码基因 ,即无鞭毛体蛋白的编码基因。     

墨西哥利什曼原虫环形DNA1含有编码核苷酸结合蛋白基因

目的　对扩增的墨西哥利什曼原虫环形 DNA1(CD1)部分片段进行测序 ,确定具有蛋白编码功能的开放阅读框。　方法　通过脉冲电泳方法分离并使用琼脂糖酶方法回收 CD1,酶切的 CD1片段克隆于 p Zero载体 ,用 M13通用引物自动测序决定核苷酸序列 ,用 GCG- PCGENE程序分析序列。　结果　测出 4385个核苷酸序列 ,通过分析发现两个开放阅读框具有蛋白质编码功能 (编码核苷酸结合蛋白 )。　结论　墨西哥利什曼原虫 CD1遗传成分含有两个编码核苷酸结合蛋白基因     

杜氏利什曼原虫激活蛋白激酶C受体的基因克隆化与序列分析

目的　克隆杜氏利什曼原虫 (LeishmaniadonovaniLd) 1S株激活蛋白激酶C受体 (RACK ,receptorofactivatedpro teinCkinase)的编码基因 ,为应用这种编码T细胞抗原的基因进行基因疫苗的研究奠定基础。方法　体外培养杜氏利什曼原虫 1S株无鞭毛体 ,常规方法提取制备基因组DNA。以硕大利什曼原虫 (Leishmaniaamjor)的RACK基因的核苷酸序列为参照 ,设计并合成利什曼原虫RACK基因序列特异性的引物。以杜氏利什曼原虫的基因组DNA为模板 ,利用多聚酶链反应(PCR)技术 ,扩增获得了杜氏利什曼原虫RACK的全长编码基因。结果　基因序列测定结果表明 ,杜氏利什曼原虫 1S株RACK基因序列长度为 981bp ,开放读码框架由 831bp组成 ,编码产物为 2 76个氨基酸残基。获得的杜氏利什曼原虫 1S株的RACK基因与来源于硕大利什曼原虫的RACK基因序列同源性达 98% (2 6 4 / 2 6 7)。结论　本研究克隆了杜氏利什曼原虫的RACK基因 ,为应用诱导T细胞免疫应答抗原的编码基因进行杜氏利什曼原虫的基因疫苗研究奠定了基础     

杜氏利什曼原虫无鞭毛体蛋白基因真核表达重组质粒构建

目的 构建杜氏利什曼原虫无鞭毛体蛋白（amastin）编码基因的真核表达重组质粒pcDNA3.1-amastin。方法 提取杜氏利什曼原虫基因组DNA进行PCR扩增,将扩增出的无鞭毛体蛋白基因片段导入质粒载体pcDNA3.1（＋）,构建真核表达重组质粒pcDNA3.1-amastin。结果 扩增出大小约550bp的无鞭毛体蛋白基因;重组质粒pcDNA3.1-amastin经鉴定正确。结论 成功构建杜氏利什曼原虫无鞭毛体蛋白基因真核表达重组质粒pcDNA3.1-amastin。     

杜氏利什曼原虫蛋白磷酸酶-2C的基因克隆化与序列分析

目的　克隆杜氏利什曼原虫 (Leishmaniadonovani,Ld) 1S株蛋白磷酸酶 2C(PP2C)的编码基因 ,为应用这种编码T细胞抗原的基因进行基因疫苗研究奠定基础。方法　体外培养杜氏利什曼原虫 1S株无鞭毛体 ,常规方法从虫体提取制备基因组DNA。以夏科氏利什曼原虫 (Leishmaniachagasi,Lc)的PP2C基因序列为参照 ,设计并合成利什曼原虫PP2C基因序列特异性的引物。结果　以杜氏利什曼原虫的基因组为模板 ,利用多聚酶链反应 (PCR)技术 ,扩增获得了杜氏利什曼原虫PP2C的全长编码基因。基因序列测定结果表明 ,杜氏利什曼原虫 1S株PP2C基因序列长度为 1317bp ,开放读码框架由 12 2 1bp组成 ,编码产物为 40 6个氨基酸残基。获得的杜氏利什曼原虫 1S株的PP2C基因与来源于夏科氏利什曼原虫的PP2C氨基酸残基序列的同源性为 95 % (387/ 40 6 )。结论　本研究克隆了杜氏利什曼原虫的PP2C基因 ,为应用诱导T细胞免疫应答抗原的编码基因进行杜氏利什曼原虫的基因疫苗研究奠定了基础     

杜氏利什曼原虫蛋白鳞酸酶—2C的基因克隆化与序列分析

目的 克隆杜氏利什曼原虫（Leishmania donovani,Ld)1S株蛋白磷酸酶2C（PP2C）的编码基因，为应用这种编码T细胞抗原的基因进行基因疫苗研究奠定基础。方法 体外培养杜氏利什曼原虫1S株无鞭毛体，常规方法从虫体提取制备基因组DNA。以夏科氏利什曼原虫（Leishmania chagasi,Lc)的PP2C基因序列为参照，设计并合成利什曼原虫PP2C基因序列特异性的引物。结果 以杜氏利什曼原虫的基因组为模板，利用多聚酶链反应（PCR）技术，扩增获得了杜氏利什曼原虫PP2C的全长编码基因。基因序列测定结果表明，杜氏利什曼原虫1S株PP2C基因序列长度为1317bp，开放读码框架由1221bp组成，编码产物为406个氨基酸残基。获得的杜氏利什曼原虫1S株的PP2C基因与来源于夏科氏利什曼原虫的PP2C氨基酸残基序列的同源性为95％（387／406）。结论 本研究克隆了杜氏利什曼虫的PP2C基因，为应用诱导T细胞免疫应答抗原的编码基因进行杜氏利什曼虫的基因疫苗研究奠定了基础。     

不同种（株）利什曼原虫毒力相关基因的表达差异

目的 观察不同种（株）利什曼原虫前鞭毛体和无鞭毛体的毒力相关基因表达情况。 方法 制备杜氏利什曼原虫、婴儿利什曼原虫、热带利什曼原虫、硕大利什曼原虫和墨西哥利什曼原虫等5种7株利什曼原虫前鞭毛体和无鞭毛体的总RNA,采用半定量RT-PCR法,以α-微管蛋白基因和3-磷酸甘油醛脱氢酶基因（GAPDH）作为阳性对照,根据GenBank公布的GDP甘露糖焦磷酸酶基因（GDPMP）、A2抗原相关蛋白基因（A2rel）、脂磷酸多糖合成蛋白1基因（LPG1）、脂磷酸多糖合成蛋白2基因（LPG2）、动基体膜蛋白11基因（KMP-11）、胱氨酸蛋白酶C基因（CPC）、亲水性酰化表面蛋白B1基因（HASPB1）、胱氨酸蛋白酶2基因（CPB2）、胱氨酸蛋白酶B2.8基因（CPB2.8）和热激蛋白100基因（CLP b）等毒力相关基因的核苷酸序列,设计特异性引物进行RT-PCR扩增,分析以上各基因在各种（株）前鞭毛体和无鞭毛体中的表达情况。 结果 各毒力基因在不同种（株）利什曼原虫的前鞭毛体和无鞭毛体中的表达明显不同,HASPB1基因在7个种（株）利什曼原虫的无鞭毛体和杜氏利什曼原虫前鞭毛体中均表达,GDPMP、LPG1、LPG2、CPB2.8、CPB2、A2rel和CLP基因分别在特定种（株）的前鞭毛体和/或无鞭毛体中表达,CPC基因仅在杜氏利什曼原虫SC10株和硕大利什曼原虫无鞭毛体内表达,KMP-11基因在7个种（株）利什曼原虫前鞭毛体或无鞭毛体内均不表达。 结论 毒力相关基因的表达存在种特异性和期特异性。     

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.