冠心病新致病基因MEF2A第一、第八外显子突变的检测

目的研究冠心病新致病基因MEF2A在中国人群突变情况。方法利用聚合酶链反应-单链构象多态性(polym erase chain reaction-single strand conform ation polymorph ism,PCR-SSCP)和DNA测序技术对156例冠心病(CAD)患者第1外显子及第8外显子进行基因突变检测。结果MEF2A基因第1外显子区域6例患者SSCP泳动异常,第8外显子区域7例患者SSCP泳动异常,但DNA直接测序未发现MEF2A基因第1、8外显子区域基因突变。结论冠心病患者在MEF2A基因第1、8外显子未发现新的突变,PCR-SSCP结果与DNA测序结果并非平行关系,SSCP同样存在假阳性。     

小鼠肿瘤线粒体DNA突变研究

目的探讨线粒体DNA(mtDNA)突变与肿瘤发生发展的关系。方法采用聚合酶链反应(PCR)技术结合限制性片段长度多态性分析(PCR—RFLP)和单链构象多态性分析(PCR—SSCP)技术了解Lewis肺癌、LA795、Hca—F、Hca—P、CT26、SCC891共6个小鼠肿瘤细胞系mtDNA的基因变异。结果经PCR—RFLP分析发现,这些肿瘤细胞系mtDNA编码区的tRNA^lle Glu Met及ND1基因核酸序列,在27个限制性内切酶酶切位点上均无差异。而在非编码区(D-loop),与对照小鼠相比,Hca—F出现了Hinf Ⅰ的新酶切位点;用PCR—SSCP分析方法对这些肿瘤细胞系mtDNA的D—loop的5’及3’端作进一步分析,在6个肿瘤细胞系中,有4个在mtDNA非编码区上存在着突变。结论D—loop是肿瘤细胞mtDNA突变的高发区,mtDNA突变在肿瘤的发生发展中可能起作用。     

不同流行区间日疟原虫疫苗候选抗原pvama1 基因DⅠ结构域的种群结构分析

目的:明确中缅边境流行区间日疟原虫(P郾vivax)pvama1 基因DomainⅠ(DⅠ)区域的多态性和种群结构。 方法:提取16 例中缅边境P.vivax 感染患者血gDNA;PCR 和测序检测其pvama1 基因DⅠ区域,参考韩国、泰国和巴布新几内 亚(PNG)流行区相应序列,采用DnaSP6-10.1 和Structure 软件进行多态性、中性检验和种群结构分析。结果:成功扩增16 例 中缅边境pvama1 基因DⅠ区;各流行区pvama1 基因的DⅠ区仔值为0.013 ~0.021,且dN / dS >1(P<0.05)。PNG 流行区Tajima‘s D* 值显著大于1(P<0.05);各流行区pvama1 基因分为3 个亚群(K=3)。结论:Pvama1 基因DⅠ区具有低多态性并经历阳 性选择,提示其为红内期疫苗候选靶位;中缅边境、泰国和PNG 流行区具有相似的种群结构,提示以其主要单倍体型为基础设 计的疫苗具有广泛的保护作用。     

RVVC患者阴道念珠菌菌种分布及优势种白念珠菌基因型分析

外阴阴道念珠菌病(vulvovaginal candidiasis,VVC)是一种由念珠菌(Candida spp.)引起的阴道黏膜感染性疾病.约75%的育龄妇女一生中发病一次,其中5%以上的患者发展为复发性外阴阴道念珠菌病(recurrent vulvovaginal candidiasis, RVVC) ~([1]).     

家族性帕金森病α-共核蛋白基因第3、4外显子的初步研究

目的 分析家族性帕金森病与α-共核蛋白基因的相关性，在其第3、4外显子中寻找相关突变或多态。方法 收集中国帕金森病家系，采用单链构象多态性(single strand conformational polymorphism，SSCP)与异源双链(heteroduplex analysis，HA)分析相结合的方法，筛查α-共核蛋白第3、4外显子中是否存在致病性突变。结果 SSCP和HA分析第3、4外显子未见单双链泳动异常。基因测序发现第4内含子5′端的第23位和67位分别插入1个c和t。结论 (1)α-共核蛋白基因的第3、4外显子不是中国家族性帕金森病的突变热点；(2)发现α-共核蛋白基因第4内含子在不同人群中有两个多态位点(23ins c和67ins t)。     

聚合酶链反应-单链构象多态性分析在家族性高胆固醇血症患者家系调查中的应用

目的　探讨聚合酶链反应 -单链构象多态性分析 (polymerase chain reaction- single strainconformation polymorphism,PCR- SSCP)在家族性高胆固醇血症 (familial hypercholesterolemiac,FH)患者家系分析中的应用价值。方法　对于经 PCR- SSCP筛查、DNA序列分析证实的 4例 FH患者 (1例纯合子 FH外显子 7发生点突变 ,1例杂合子点突变位于外显子 14,2例杂合子点突变位于外显子 4的 3′部分 ) ,用 PCR- SSCP分析各家系成员共 2 3例 ,并对基因型和表型进行比较。结果　各家系成员均从基因水平明确了诊断 ,2 3例个体中 ,除先证者外 ,还发现 1例纯合子 ,8例杂合子。结论　 PCR- SSCP可对 FH先证者家系进行分析 ,并对其家系成员早期诊断 ,以便提供咨询和指导。     

粘多糖贮积症Ⅱ型患者IDS基因的一个新突变

目的　研究粘多糖贮积症 型 ( mucopolysaccharidosis type ,MPS )患者的艾杜糖 - 2 -硫酸酯酶 ( iduronate- 2 - sulfatase,IDS)基因的基因突变。方法　应用聚合酶链反应 -单链构象多态性( polymerase chain reaction- single strand conformation polymorphism,PCR- SSCP)对患者的 IDS基因可能的常见突变第 2、3、5、7～ 9外显子进行检测 ,并对 PCR- SSCP检出的突变进行直接测序 ,测序发现的突变进行 PCR-限制性酶切分析验证。结果　经 PCR- SSCP和 DNA序列分析发现该患者的第 7外显子发生新的点突变 ( G12 5 3T) ;聚合酶链反应 -限制性片段长度多态性 ( PCR- RFL P)电泳检测示患者和母亲出现突变导致的酶切位点 ,进一步验证了序列分析结果。结论　筛查所得的点突变 G12 5 3T可能是该 MPS 患者的致病原因     

PAI-1启动子区4G/5G基因多态性、ACE插入/缺失多态性与脑卒中的相关性

目的探讨纤溶酶原激活物抑制物-1(PAI-1)启动子区基因多态性和血管紧张素转换酶(ACE)插入/缺失多态性与脑卒中的关系。方法 PCR检测203例脑卒中患者和139名健康对照者PAI-1基因启动子区4G/5G多态性、ACE基因插入/缺失多态性,同时应用比色法测定血清ACE活性,发色底物法测定PAI-1活性。结果脑梗死(CI)组PAI-1活性(0.769±0.163 AU/mL)、ACE活性(43.42±14.36 U/L)明显高于对照组(0.652±0.116 AU/mL和31.28±8.64 U/L,P<0.01);CI组PAI-I基因4G纯合子、ACE D/I基因DD纯合子比例明显高于对照组(P<0.01);PAI-I基因4G/4G基因型与ACE基因D/D基因型对CI发病可相互协同作用(P<0.01)。结论 PAI-1基因4G/4G基因型和ACE基因D/D基因型均可能是CI发病的危险因素,且具有协同作用。     

维吾尔族苯丙酮尿症R158Q突变基因杂合子报告

目的报道在新疆维吾尔族家系中检出的苯丙酮尿症(PKU)R158Q突变基因杂合子。方法采用PCR结合单链构象多态性(SSCP)分析技术和PCR产物直接测序方法确定PAH基因突变类型。结果分析患者及其父母PAH第7、6、11、3、12、5外显子基因,发现在外显子5中患者的SSCP电泳行为与正常对照不同而与父亲的电泳条带位置一致,测序结果显示,患者和父亲的PAH基因cDNA第473位发生了G→A点突变,为R158Q突变型杂合子。结论国内在维吾尔族中报道PKU R158Q突变基因杂合子尚属首次。     

复发性外阴阴道念珠菌病危险因素分析

目的探讨复发性外阴阴道念珠菌病（RVVC）发病的危险因素。方法采用念珠菌镜检和培养的方法将160例患者中阳性者分为外阴阴道念珠菌病（WC）组和RVVC组,分析各种危险因素在3组中的差异。结果使用抗生素、阴道冲洗、口服避孕药、妊娠与RVVC的发生有关。结论使用抗生素、阴道冲洗、口服避孕药、妊娠是RVVC的危险因素。     

Single-strand conformation polymorphism of microsatellite for rapid strain typing of Candida albicans.

Single-strand conformation polymorphisms (SSCP) of Candida albicans' microsatellite CAI were characterized. Among the 76 clinical isolates recovered from different patients (independent strains), 60 distinct CAI SSCP patterns were recognized, resulting in a discriminatory power of 0.993. The multiple isolates recovered sequentially from the same or different body locations of the same patient showed exactly the same CAI SSCP pattern. The reliability of the SSCP analysis was confirmed by GeneScan and sequence analyses. From the same set of independent strains, 59 distinct CAI genotypes were identified by GeneScan analysis. Sequence comparison showed the advantage of SSCP over GeneSan analysis in the detection of point mutations in the microsatellite. The results indicated that PCR SSCP analysis of CAI microsatellite is a powerful and economical approach for rapid strain typing of C. albicans in clinical laboratories, especially in the detection of microevolutionary changes in microsatellites and in large-scale epidemiological investigation.     

白色念珠菌性阴道炎对人精子顶体反应影响机制的初步探讨

目的探讨白色念珠菌性阴道炎对人精子顶体反应（AR）的影响机制。方法以ELISA和硝酸还原酶法分别检测阴道分泌物中的IL-8与NO,采用BAEE/ADH法测定顶体酶的活性,并通过FITC-PSA法检测AR。结果无症状的白色念珠菌携带者阴道分泌物中IL-8及NO水平无显著变化,但在白色念珠菌性阴道炎患者阴道分泌物中,其水平显著升高,且二者水平呈正相关;IL-8对精子顶体酶活性和AR均无影响;低浓度的NO可诱导精子顶体酶活性,同时促进AR,而高浓度的NO对其起抑制作用。结论白色念珠菌性阴道炎患者阴道分泌物IL-8和NO水平升高,可直接或/和间接抑制精子顶体酶活性和AR,其中高浓度的NO起着直接的抑制作用,而IL-8可能间接参与了此作用。     

Electric and chemical fusions for the production of monoclonal antibodies reacting with the in-vivo growth phase of Candida albicans

To obtain monoclonal antibodies (MAbs) directed preferentially against the pathogenic phase of Candida albicans, mice were immunised with germ tubes of C. albicans serotype A, strain VW.32, killed by exposure to ultraviolet (UV) irradiation. Fusions were performed either by the standard chemical procedure with polyethylene glycol, or by electric discharge following linkage of the myeloma and lymphocyte cells with a Concanavalin A-mannoprotein bridge. The preliminary characteristics of one MAb obtained from each of these fusions are described. An IgM antibody (3B7) obtained from the chemical fusion reacted with a polysaccharide antigen that was heterogeneously distributed on both in-vitro and in-vivo forms of C. albicans. This MAb agglutinated different strains of C. albicans irrespective of their serotype. An IgG1 antibody (3G6) that had been obtained from the electric fusion was found to react in vitro with a proteinaceous antigen located only on the germ tubes of strain VW.32. However, MAb 3G6 displayed strong reactivity against all growth forms of C. albicans in vivo and reactivity extended to other strains.     

Immunoblot analyses of Candida albicans-associated antigens and antibodies in human sera.

We tested 10 patient sera for the presence of immunoglobulin G (IgG) antibodies to Candida albicans and for C. albicans antigens by immunoblot analysis (i.e., electrotransfer blot radioimmunoassay) (G. E. Smith and M. D. Summers, J. Virol. 39:125-137, 1981). We evaluated sera from two patients at risk for candidiasis, five patients with systemic candidiasis documented by culture, and two patients who had experienced transient candidemia. Both the specificity and the relative amount of IgG antibodies to C. albicans in each serum sample were readily visualized by this technique, as was the absence of antibody from serum of neonatal and immunocompromised patients. No antibody species appeared to be uniquely associated with candidiasis patients (i.e., each antibody species present in the candidiasis patient was also present in sera of normal individuals or "at-risk" patients). IgG from rabbits immunized with whole cells or with a cytoplasmic fraction of C. albicans was used to detect C. albicans antigens in patient sera. Although several antigens were detected in the sera from patients with candidiasis, the same antigens were also detected in sera from patients at risk and in normal human serum. No antigens were detected in human serum when preimmune rabbit sera were used. These results suggest that the antigens detected by the rabbit antisera were human serum proteins that cross-reacted with C. albicans antigens. These findings may have important implications in studies of both the pathobiology of C. albicans and the serodiagnosis of candidiasis.     

Mutational analysis of the Cu/Zn superoxide dismutase gene in 23 familial and 69 sporadic cases of amyotrophic lateral sclerosis in Belgium.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder caused by degeneration of motor neurons of the spinal cord and brain. The majority of ALS cases are sporadic (SALS). However, in 10-15% of ALS cases the disease is inherited as an autosomal dominant trait (familial ALS or FALS). We used a non-radioactive SSCP method, in combination with solid phase sequencing, to screen the entire SOD1 (Cu/Zn superoxide dismutase) coding region and flanking intronic sequences for mutations. Twenty-three patients from 11 ALS families and 69 SALS patients of Belgian origin were studied. Three different mutations were identified (L38V, D90A and G93C) in seven families. Importantly, the D90A was only found in the heterozygous state. In addition two single base pair variants (IVS1 + 19G > A and AAC139 AAT) were identified in two SALS patients. These results suggest that the SOD1 analysis is useful in FALS but less so in SALS cases. The SSCP analysis has proved fast and reliable for this purpose.     

Analysis of the preS1 gene of hepatitis B virus (HBV) to define epidemiologically linked and un-linked infections in South Africa

Summary.  Analysis of the preS1 gene of hepatitis B virus was used to define two nosocomial outbreaks of HBV infection. In an outbreak in an oncology unit we had previously shown by single stranded conformational polymorphism (SSCP) analysis of a 189 bp fragment of the preS1 gene, that 52 children were infected with HBV strains that displayed only 5 different SSCP profiles. Sequencing of a 383 bp fragment encompassing the entire preS1 gene, revealed that isolates with same SSCP profile were identical in sequence across the entire preS1 gene, confirming that those patients with the same SSCP pattern had epidemiologically linked infections. A second outbreak involved 8 liver transplant patients from two different hospitals, 5 of whom were from the same hospital at which the oncology outbreak had occurred. Two of these 5 patients had HBV strains that were identical to strains from the oncology unit and nosocomial transmission probably accounted for the infections in these two, while diversity of both SSCP profiles and sequence data of remaining 6 patients supported the conclusion that they had not been infected from a common source. The donor liver is believed to be the most likely source of infection in these patients. HBV isolates from patients infected in the community were used as a standard for the general degree of preS1 sequence variation of local HBV strains. Phylogenetic analysis and comparison with reference HBV clones revealed that of 27 local HBV strains, genotypes A and D occurred most frequently and were identified in 14 and 12 patients respectively, while genotype C was detected in one patient. Received January 27, 1997 Accepted April 9, 1997     

Composition, antifungal and radical scavenging activities of 4 propolis

HPLC/MS analysis revealed that the main constituents of Brazilian propolis A and B were artepillin C and drupanin, while those of New Zealand propolis C were pinocembrin, galangin and alkylphenol. No flavonoid or phenolic acid was detected in Japanese propolis D. Propolis C showed comparatively potent activity against growth of Trichophyton mentagrophytes, against filament formation of Candida albicans and potent scavenging activity against 1,1-diphenyl-2-picrylhyrazyl radical, but was less active against growth of C. albicans, as compared with those of thyme thymol essential oil, which was used as a positive control. Propolis A, B, and D were weak in antifungal activity, but showed more potent radical scavenging activity than thyme thymol oil. These results reveal the unique bioactivity of propolis, suggesting a possible application for antifungal therapy.     

Molecular characterization of new clinical isolates of Candida albicans and C. dubliniensis in Japan: analysis reveals a new genotype of C. albicans with group I intron.

The genetic diversity of recent clinical isolates of Candida albicans in Japan was studied on the basis of amplified DNA band lengths determined with a specific PCR primer reported to have been designed to span a transposable intron region in the 25S rRNA gene. Our analyses of 301 clinical isolates of C. albicans showed that they could be classified into five genotypes: genotype A (172 isolates), genotype B (66 isolates), genotype C (56 isolates), genotype D (C. dubliniensis; 5 isolates), and a new genotype (designated genotype E; 2 isolates). The new genotype E was characterized to have a group I intron-like sequence, which is longer than hitherto reported ones and which has a nucleotide sequence length of 962 bp. Our analysis of the 962-bp sequence indicated that it is composed of an intron similar to that of C. dubliniensis of 621 bp with a 341-bp insertion. Analysis of the sequence of the internal transcribed spacer (ITS) region of the genotype E strain showed that its sequence is identical to those of strains of other genotypes, with only a few base substitution differences. Throughout the study, the possible horizontal transfer of the group I intron between C. dubliniensis and C. albicans was suggested. A high degree of correlation between the presence of a group I intron in C. albicans genotype E and susceptibility to the antifungal agent flucytosine was observed. The five isolates of C. dubliniensis examined in the present study showed genetic diversity when they were compared by randomly amplified polymorphic DNA fingerprinting pattern analysis, and this diversity was also confirmed by the analysis of ITS region sequences.     

Genotypic profiles and virulence attributes of Candida albicans isolates from patients with oral lichen planus

Candida albicans carriage has been found to be increased in patients with oral lichen planus. In the present work we have investigated the genotypic profiles of 112 C. albicans strains isolated from patients with erosive or nonerosive OLP, and from healthy controls. The virulence attributes of the isolated strains were compared to elucidate the pathogenetic mechanisms through which C. albicans may cause OLP. We have characterized the genotypic profiles of these isolated strains using a randomly amplified polymorphic DNA assay. In addition, we used assays to measure adhesion to buccal epithelial cells and phospholipase activity to evaluate the virulence attributes of these isolates. Our RAPD analyses revealed four different genotypes, named type A to D, among all isolates, and identified statistically significant associations with disease conditions. Specifically, type A (58.1%) and C (29.0%) were primarily found in erosive OLP, while type A (33.3%) and D (58.3%) were identified in nonerosive OLP. However, the healthy group seemed to have type B (38.5%) and D (61.5%). Using adhesion to BEC assay, we demonstrated that the isolates with genotype A had the strongest adherence among the four genotypes (P=0.000<0.05). The phospholipase activity of the isolates with genotype A and C was higher than for those with genotype B and D (P=0.000<0.05). In conclusion, some C. albicans isolates with special genotypic profiles and virulence attributes may contribute to the pathogenesis of OLP.     

A novel nonsense mutation at Glu-631 in a Spanish family with complement component 7 deficiency

Deficiency of the seventh component of complement (C7D) is frequently associated with recurrent neisserial infections. We report in the present study the genetic basis for C7D in a Spanish family. We used exon-specific polymerase chain reaction (PCR)/single-strand conformation polymorphism (SSCP) analysis as a screening step for mutations, followed by direct sequencing of the target exon. The mutation in the proband was a homozygous G-to-T transversion at nucleotide 1957, the first nucleotide of the codon GAG for Glu-631, leading to a stop codon TAG (E631X). Our result provides further evidence that the molecular pathogenesis of C7D is heterogeneous. Received: October 20, 1998 / Accepted: December 25, 1998