散发性扩张型心肌病相关TBX20基因突变谱分析

目的:分析散发性扩张型心肌病(dilated cardiomyopathy,DCM)相关TBX20基因突变谱。方法:从中国汉族人群中选择105例散发性DCM患者和130名健康对照者,采集静脉血并使用核酸纯化试剂盒纯化基因组DNA,采用PCR试剂扩增TBX20基因的全部编码外显子,对扩增的TBX20基因片段进行测序,将所得序列与GenBank数据库中的TBX20基因序列进行对比,借助MUSCLE分析突变氨基酸在进化上的保守性,应用MutationTaster和PolyPhen-2预测突变的致病性。结果:在1例散发性DCM患者识别出了1个新的杂合性TBX20基因变异c.832GA,即p.D278N突变,但该突变在130名对照者和其他散发性DCM患者中均未检出。跨物种TBX20蛋白序列比对分析表明,第278位的天冬氨酸在进化上保守,该TBX20基因突变具有致病性。结论:发现散发性DCM相关TBX20基因新致病性突变c.832GA,将有助于DCM患者的遗传咨询和个体化处理。     

特发性房颤相关GATA5基因新突变的识别

目的:识别特发性房颤(AF)相关GATA5基因新突变. 方法:收集120例无血缘关系的特发性AF患者和200名无血缘关系且种族匹配的健康对照者的临床资料和血标本.抽提基因组DNA,通过聚合酶链反应扩增AF候选基因GATA5的全部外显子及其两侧的部分内含子,采用双脱氧核苷链末端合成终止法对全部扩增片段进行测序.将所测的序列与GenBank数据库中的GATA5基因序列进行比对,以发现GATA5基因突变.应用多序列比对软件ClustalW评估突变氨基酸的保守性,应用致病性预测软件MutationTaster预测突变的致病性. 结果:在2例AF患者各发现1个新的GATA5基因杂合错义突变,突变率约为1.67％.突变分别为GATA5基因编码核苷酸序列第668位的腺嘌呤(adenine,A)变为胸腺嘧啶(thymine,T),即c.668A＞T突变；第863位的A变为胞嘧啶(cytosine,C),即c.863A＞C突变.多序列比对显示2种突变氨基酸在进化上均高度保守,致病性预测表明2种突变均有致病性. 结论:本研究识别出AF相关GATA5基因新突变,有助于揭示AF发生的分子机制.     

孤立性房颤相关SCN4B基因突变谱分析

目的：发现孤立性房颤相关SCN4B基因新突变. 方法：收集160例孤立性房颤患者和200名健康对照者的临床资料和血标本,抽提基因组DNA.通过聚合酶链反应扩增房颤候选基因SCN4B的编码区和剪接位点,采用双脱氧核苷链末端合成终止法测序以发现SCN4B基因突变.应用ClustalW软件评估突变氨基酸的保守性,应用MutationTaster软件预测突变的致病性. 结果：在2例孤立性房颤患者各发现1个新的SCN4B基因杂合错义突变,突变率为1.25％.其中1个是SCN4B基因编码核苷酸序列第409位的腺嘌呤（adenine,A）变为鸟嘌呤（guanine,G）,即c.409A＞G突变；另一个是SCN4B基因编码核苷酸序列第511位的G变为A,即c.511G＞A突变.多序列比对显示2种突变氨基酸在进化上均高度保守.致病性预测表明2种突变均有致病性.结论：本研究发现孤立性房颤相关SCN4B基因新突变,有助于揭示房颤新的分子机制.     

特发性扩张型心肌病相关LRRC10基因新突变的识别

目的:识别特发性扩张型心肌病(dilated cardiomyopathy,DCM)相关LRRC10基因新突变。方法:收集120例特发性DCM患者和200名健康对照者的血标本,用基因组纯化试剂盒提取基因组DNA。通过聚合酶链式反应扩增LRRC10基因的编码外显子和其两侧的内含子,在DNA测序仪上对扩增片段进行测序。将所测序列与核苷酸数据库中的LRRC10基因序列进行比对,寻找可能的基因突变。借助在线程序MUSCLE分析突变氨基酸的保守性,应用MutationTaster和PolyPhen-2预测突变的致病性。结果:在1例特发性DCM患者中发现了1种新的LRRC10基因杂合错义突变,即p.R157G突变,突变率约为0.83%。在200名健康对照者无此基因突变。多物种LRRC10蛋白的氨基酸序列对比显示,第157位的精氨酸在进化上完全保守,致病性预测表明这种突变具有致病性。结论:本研究发现特发性DCM相关LRRC10基因新突变,揭示了特发性DCM新的分子病因。     

先天性心脏病相关GATA5基因突变研究

目的:识别先天性心脏病(CHD)相关GATA5基因新突变。方法:收集100例无血缘关系的CHD患者和200名无血缘关系且种族匹配的健康对照者的临床资料和血标本。抽提基因组DNA,通过聚合酶链反应扩增GATA5基因的编码外显子及其剪接位点,采用双脱氧核苷链末端合成终止法对全部扩增片段进行测序。将所测的序列与GenBank数据库中的GATA5基因序列进行比对以发现GATA5基因突变。应用多序列比对软件ClustalW评估突变氨基酸的保守性,应用致病性预测软件MutationTaster预测突变的致病性。结果:在2例CHD患者各发现1个新的GATA5基因杂合错义突变,突变率为2%。其中1个是GATA5基因编码核苷酸序列第395位的鸟嘌呤(guanine,G)变为胸腺嘧啶(thymine,T),即c.395G>T突变;另一个是GATA5基因编码核苷酸序列第991位的胞嘧啶(cytosine,C)变为腺嘌呤(adenine,A),即c.991C>A突变。多序列比对显示2种突变氨基酸在进化上均高度保守。致病性预测显示2种突变均有致病性。结论:本研究发现了CHD相关GATA5基因新突变,有助于揭示CHD新的分子机制。     

先天性心脏病相关PITX2c基因新突变研究

目的:研究先天性心脏病(congenital heart disease,CHD)相关PITX2c基因的新突变。方法:收集150例CHD患者和200名正常对照者的外周静脉血标本,使用DNA纯化试剂盒分离基因组DNA。使用DNA聚合酶扩增PITX2c基因的编码区和剪接位点,应用DNA测序试剂盒在DNA分析仪上对扩增片段进行测序。将所测序列与GenBank数据库中的PITX2c基因序列进行比对以发现PITX2c基因突变。使用在线程序MUSCLE分析突变氨基酸的保守性,分别应用MutationTaster和PolyPhen-2分析突变氨基酸的致病性。结果:在1例散发性CHD患者发现了1个新的PITX2c基因杂合错义突变,即p.S101G突变,突变率约为0.67%。该错义突变不存在于200名正常对照者。跨物种PITX2c蛋白之氨基酸序列对比显示第101位的丝氨酸在进化上完全保守,致病性预测显示所发现的PITX2c基因变异是致病性突变。结论:本研究揭示了CHD相关PITX2c基因新突变,对于制定新的CHD防治策略具有潜在的意义。     

特发性扩张型心肌病相关GATA6基因新突变的识别

目的:识别特发性扩张型心肌病(DCM)相关GATA6基因新突变。方法:入选132例特发性DCM患者和220名健康对照者,收集其临床资料和外周静脉血,使用DNA纯化试剂盒抽提基因组DNA,采用聚合酶链反应试剂扩增GATA6基因的整个编码区,应用循环测序试剂盒对扩增的GATA6基因片段进行测序,将所测序列与Nucleotide数据库中的GATA6基因序列进行对比分析以识别GATA6基因突变。应用MUSCLE和MutationTaste在线系统评估突变氨基酸进化上的保守性和突变的致病性。结果:通过序列分析发现1例散发性DCM患者的GATA6基因新变异c.1165GT,相当于p.E389X突变,在220名健康对照者中没有检测出该无义突变。跨物种GATA6蛋白序列比对分析表明,被改变的氨基酸在物种进化上完全保守,致病性预测显示该GATA6基因突变具有致病性。结论:发现散发性DCM相关GATA6基因新突变,扩大了DCM相关GATA6基因突变谱。     

先天性心脏病相关HAND1基因新突变研究

目的:研究先天性心脏病(congenital heart disease,CHD)相关HAND1基因新突变。方法:入选CHD患儿136例及健康对照儿童200名,抽提基因组DNA,通过聚合酶链反应测序筛查HAND1基因突变。应用计算机软件评估突变氨基酸的保守性并预测突变的致病性,应用双荧光素酶报告基因分析系统分析突变对HAND1功能的影响。结果:在1例法洛四联征患儿发现1种新的HAND1基因杂合突变,其编码核苷酸序列第389位的胸腺嘧啶突变为鸟嘌呤(c.389TG),相应氨基酸序列的第130位亮氨酸变为精氨酸(p.L130R)。该突变改变了进化上保守的氨基酸序列并被预测为有致病性,功能研究揭示突变型HAND1的转录激活功能显著降低。结论:该HAND1基因功能缺失性新突变可能是CHD的少见分子病因。     

SHOX2基因与特发性心房颤动的关系研究

目的:研究SHOX2基因与特发性心房颤动(IAF)的关系。方法:收集178例汉族IAF患者和218名匹配的健康对照者。通过聚合酶链反应-DNA测序分析SHOX2基因的编码外显子及剪接序列。将测出的序列与核苷酸数据库中的SHOX2序列进行对比,查找SHOX2基因突变。通过在线计算机程序MUSCLE评估突变氨基酸进化上的保守性,应用PolyPhen-2、MutationTaster和PROVEAN软件分析SHOX2基因突变的致病性。结果:在1例IAF患者中发现SHOX2基因新突变,c.632GC (p.Trp211Ser),该错义突变不存在于218名健康对照者中,多物种SHOX2蛋白序列比对分析显示被改变氨基酸在进化上完全保守,致病性预测表明所识别的SHOX2基因突变具有致病性。结论:c.632GC是基因新突变,对IAF的早期防治具有潜在的临床意义。     

儿童先天性心脏病TBX20基因突变及功能探讨

目的：对先天性心脏病（先心病）患儿进行TBX20基因突变研究,探讨TBX20基因与先心病发生机制之间的关系。方法：对203例中国汉族先心病患儿（病例组）进行TBX20基因编码区测序,同期300例门诊体检健康儿童为对照组,检测可能的先心病相关性基因突变。对新发现的突变应用蛋白质结构预测服务器I-TASSER进行同源建模,对TBX20基因野生型及突变后的蛋白质结构模型进行预测分析。并进一步构建TBX20野生型及突变型表达载体,分别转染到COS7细胞中,通过双荧光素酶报告基因检测它们在NKX2.5、GATA4协同作用下对下游靶基因心钠素（ANF）启动子转录激活作用的影响。结果：（1）病例组1例房间隔缺损患儿发现一个新的TBX20基因错义突变c.187G>A(A63T),DNA序列中第187位鸟嘌呤突变为腺嘌呤,导致氨基酸序列中第63位丙氨酸变为苏氨酸,在对照组中均未发现该变异。（2）对错义突变A63T进一步研究中,TBX20同源建模显示第63位的非极性、疏水性丙氨酸变为极性、亲水性的苏氨酸,与Gly65、Gly68间的氢键断裂,与Asp62、His64形成新的氢键,导致蛋白质构象发生变...     

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.