Phylogeny of Apaturinae Butterflies (Lepidoptera: Nymphalidae) Based on Mitochondrial Cytochrome OxidaseⅠ Gene

The phylogenetic relationships of genera in the subfamily Apaturinae were examined using mtDNA sequence data from 1,471 bp of cytochrome oxidase subunit Ⅰ （COI）. The mitochondrial COI gene from a total of 16 species in 11 genera were sequenced to obtain mtDNA data, along with those of 4 species obtained from GenBank, to construct the MP and the NJ trees using Athyma jina, Penthema adelma, Polyura nepenthes, and Charaxes bernardus as outgroups. The transitions at the third codon positions of the COI data set were found saturated, but they were retained for analysis, because they contain the majority of the phylogenetic information. The impacts of equal weight assumptions for all characters in the parsimonious analysis were assessed by potential alternations in clades in response to different transition/transversion weighting schemes. The results indicated four distinct major groups in Apaturinae. Moreover, several well supported and stable clades were found in the Apaturinae. The study also identified undetermined taxon groups whose positions were weakly supported and were subject to changes under different weighting schemes. Within the Apaturinae, the clustering results are approximately identical to the classical morphological classification. The mtDNA data suggest the genus Mimathyma as a monophyletic group. Lelecella limenitoides and Dilipa fenestra have close relationship with very strong support in all phylogenetic trees. It also supports the taxonomic revision of removing several species from Apatura to other genera, namely Mimathyma schrenckii, M. chevana, M. nycteis, Chitoria subcaerulea, C. fasciola, C. pallas, and Helcyra subalba.

Development and Clinical Application of a Single-tube Nested PCR Method to Amplify the DNA Polymerase Ⅰ Gene of Treponema Pallidum

Objective: To develop a sensitive, specific and simple method for detection of extremely low numbers of T. pallidum in clinical specimens, as a significant addition to the serologic tests for syphilis diagnosis. Methods: Double-tube nested PCR(DN-PCR) and single-tube nested PCR(SN-PCR) assays were performed to amplify specific fragments of the DNA poly-merase I gene(polA) of T. pallidum. Sensitivity and specificity of the two PCR assays were tested. Eighty-six whole blood specimens from persons with suspected syphilis were detected by the two nested PCR methods. The TPPA test was used as a comparison for detecting syphilis in sera from corresponding patients. Results: Only specific amplicons could be obtained during amplification of the T. pallidum polA gene and the detection limit was approximately 1 organism when analyzed on gel by the two PCR methods. Of 86 clinical specimens, 62 were positive by TPPA. Of these, 54 and 51 were positive by the DN-PCR and SN-PCR, respectively, which does not represent a statistically significant difference between the two PCR tests. Of 24 TPPA-negative specimens, 5 were positive by both DN-PCR assay and SN-PCR assay. Conclusion: The SN- polA PCR method is extremely sensitive, specific and easy to perform for detecting low numbers of T. pallidum in clinical blood specimens as a complementary to serology for syphilis diagnosis.

Analysis of Rb gene Xba Ⅰ polymorphism in Shaanxi aged atherosclerosis population

Objective:To investigate variable number tandem repeat (VNTR) polymorphism of the 17th intron of Rb gene in Shaanxi aged population and the relationship between the polymorphism of Rb gene and atherosclerosis(AS) genetic suscepti- bility. Methods: VNTR polymorphism of the 17th intron of Rb gene were examined in 100 Shaanxi aged AS patients and 100 Shaanxi aged control individuals by PCR-Rb-Xba Ⅰ-RFLP. Results::Two alleles were found both in AS group and control group, which were separately 945 bp(S1) and 630bp + 315bp(S2). S1S2 genotype was the most frequent one in the two populations. Significant difference in allele frequency was not found between AS group and control group, and allele frequency was no significant difference between Chinese and Caucasian. Conclusion: Xba Ⅰ enzyme site of Rb gene could have been certainly stable in AS population, and it was inferred that the polymorphism locus was not liable to cause mutation, which might not implicated in the formation of AS.

The gene diagnosis and mutation survey of autosomal dominant polycystic kidney disease Ⅰ

Objective: To explore gene diagnosis of autosomal dominant polycystic kidney disease (ADPKD Ⅰ ),and to look for the typical mutation in order to improve the gene diagnosis. Metbods: southern blot and PCR wasused to observe the mutation condition of 3' end single copy region of ADPKD Ⅰ gene ; Amplifying and analyzingthe microsatellite SM7 by PCR. Results: ①After the probe AH4 was hybridized with 16 patients' genomic DNAby Southern blot, the common 15 kb fragments were found in every one; ②For 27 patients, 5. 72 kb genomicDNA, which is between the probe AH4 and JH14, was amplified by PCR, and no 5. 5 kb genomic DNA deletionwas found in this region; SM7 was amplified in lO9 health persons, its PIC was 0.76, and was closely linked towith ADPKD Ⅰ gene in three patient families. Conclusion: In Han nation, ①No large genomic DNA segmentdeletion could be found frequently in ADPKD Ⅰ gene 3' end single copy region; ②The PIC of SM7 is high, it canbe used to make rapid gene diagnosis in about 70% ~80% ADPKD Ⅰ families.

Establishment of a Real-time Fluorescent Quantitative RTPCR Method for Detecting NP Gene of Class Ⅰ Newcastle Disease Virus(NDV)

Newcastle disease( ND) is one of the most serious infectious diseases that infect the poultry industry.There is only one serotype of Newcastle disease virus( NDV),but NDVs can be divided into two distinct classes( class Ⅰ,and class Ⅱ) according to their genetic relationship.To develop a method for rapid quantitative detection of class Ⅰ NDV,a pair of primers and a TaqM an probe were designed and synthesized according to the conservative sequence of NP gene of class Ⅰ NDV.The positive recombinant plasmid harboring NP gene of JS-18-05 isolate was used as a positive template to establish the standard curve.A real-time fluorescent quantitative RT-PCR method was established for rapid detection of class Ⅰ NDV with strong specificity,high sensitivity and good repeatability.The established method exhibited a good linear relationship within the concentration of 102 to 108 copies of NDV,by which 1 μl of 10 copy of NDV nucleic acid could be detected in the initial template.Compared with conventional virus isolation methods,the established method had similar sensitivity and led to the same results in detecting33 class Ⅰ,class Ⅱ NDV isolates.The study provided the basis for rapid quantitative detection of class Ⅰ NDVs and further clarification of their pathogenicity and pathogenic mechanism in poultry.

Inhibition of α_1(Ⅰ) collagen gene in vitro transcription by antisense oligodeoxynucleotides

Objective and Methods: Excessive accumulation of collagen typeⅠ and type Ⅲ causes the formation of keloids and hypertrophic scars. To understand the mechanism by which antisense oligodeoxynucleotide (Oligo) acts on in vitro transcrption α1 (I) collagen gene, isotopes (α-32pGTP) was incorporated into 2 SP6 in vitro transcription systems. Results and Conclu- sion: Oligo 2 (at the transcription start region) could effectively inhibit in vitro transcription of pGEM3-Col13 and the control (random oligodeoxynucleotides) showed no inhibition. However, oligo 1 (at the transcription start region) obviously inhibited the in vitro transcription of pGEM3-Col14, while Oligo 2, which targeted at the down stream region (about 200 bp) of the promoter showed no significant inhibition effect.

Contruction of the Genetic Engineering Strain Expressed Nontoxic ST_1-LT_B Fusion Protein Against Enterotoxigenic Eschenichia coli

Thermostable enterotoxinⅠ(ST1) mutant genes and thermolabile enterotoxin B subunit (LTB)genes were amplified by PCR from plasmids of Eschenichia coli C83902. The recombinantexpression plasmid pZST3LTB containing ST1-LTB fusion gene was constructed by recombinantDNA technique and then transformed into Escherichia coli BL21(DE3). The ST1-LTB fusionprotein was highly expressed in recombinant strain BL21(DE3)(pZST3LTB) and the fusionprotein was about 38.53% of total cellular protein by SDS-PAGE and thin-layer gelscanning analysis. More important, mice immunized with crude preparation containing thefusion protein inclusion bodies or inactivated recombinant strain produced antibodiesthat were able to recognize ST1 in vitro. These sera antibodies were able to neutralizethe biological activity of native ST1 in the suckling mouse assay. Hence the ST1-LTBfusion protein was nontoxic and immunogenic, the constructed recombinant strain BL21(DE3)(pZST3LTB) could be used as a candidate of vaccine strain.

The Genetic Structure and Diversity of Repomucenus curvicornis Inhabiting Liaoning Coast Based on Mitochondrial COⅠ Gene and Control Region

[Object] This study was conducted to explore the genetic diversity and structure of the wild Repomucenus curvicornis inhabiting Liaoning Coast, China. [Method] The mitochondrial COⅠ gene and control region（CR） were PCR amplified from the wild R. curvicornis populations from the Liaodong Bay（n=22） and the northern Yellow Sea（n=18）, sequenced and analyzed for genetic diversity. [Result] The contents of A, T, C and G of 624 bp COⅠ gene were 24.09%, 31.04%, 25.28%, and 19.59%, and those of 460 bp CR fragment were 32.96%, 32.80%, 14.86% and 19.38%, respectively. The total number of variable sites, average number of nucleotide differences（ k）, haplotype diversity（H） and nucleotide diversity（π） based on COⅠ gene were 38, 4.67,（0.96±0.02） and（0.007 5±0.004 2）, and those based on CR fragment were 26, 3.35,（0.97 ±0.02） and（0.007 3±0.004 3）, respectively. Based on mitochondrial COⅠ gene and CR, the genetic diversity of Liaodong Bay population was lower than that of the northern Yellow Sea population. The AMOVA analysis based on CR fragments revealed almost significant genetic divergence between the Liaodong Bay and the northern Yellow Sea populations, while there was no significant genetic divergence based on COⅠ gene. The results showed that CR and COⅠ gene are effective molecular markers for detecting the genetic diversity of R. curvicornis population, while CR is more reliable than COⅠ gene in detecting the genetic structure. [Conclusion] CR is an appropriate marker for genetic analysis of marine fish population.

The Truncated Gene cfaD′ Positively Regulates CFA/Ⅰ Expression of Enterotoxigenic Escherichia coli

The gene cluster cfaABCED’ of enterotoxigenic Escherichia coli, encoding the fimbriae which is called colonization factor antigen located on a plasmid. It is positively regulated by cfaR, a member of the AraC family, and the cfaD’ gene region, which is located downstream of cfaE and is homologous to cfaR, had been described as a truncated cryptic gene. In the present study we observed that the CFA/ fimbriae subunit, cfaB, was expressed in lower amount by the cfaABCED’ clone pNTP513 in host E. coli HB101. The expression of CFA/ diminished by deletion of cfaD’ gene region from pNTP513, and was restored by acquisition of cfaD’ in trans. Furthermore, CFA/ expression by cfaD’ deletion mutant, the cfaABCE clone, was remarkably increased by the presence of cfaD’ in trans in a topoisomerase A deficient strain of E. coli DM800. These data suggest that cfaD’ region is a functional region of gene, that regulates the CFA/ expression with cfaR by unknown mechanism.

Association of Polymorphisms in Angiotensin-converting Enzyme and Type 1 Angiotensin Ⅱ Receptor Genes with Coronary Heart Disease and the Severity of Coronary Artery Stenosis

To explore the relation of angiotensin-converting enzyme （ACE） and angiotensin Ⅱ type 1 receptor （AT1R） gene polymorphism with coronary heart disease （CHD） and the severity of coronary artery stenosis, 130 CHD patients who underwent coronary angiography were examined for the number of affected coronary vessels （≥75% stenosis） and coronary Jeopardy score. The insertion/deletion of ACE gene polymorphism and AT1R gene polymorphism （an A→C transversion at nucleotide position 1166） were detected by using polymerase chain reaction （PCR） and restriction fragment length polymorphism （RFLP） in CHD patients and 90 healthy serving as controls. The resuits showed that DD genotype and of ACE were more frequent in CHD patients than that in control group （38.5% vs 14.4%, P〈0.001）. The frequency of the ATIR A/C genotypes did not differ between the patients and the controls （10% vs 13.1%, P〉0.05）. The relative risk associated with the ACE-DD was increased by AT1R-AC genotype. Neither the number of affected coronary vessels nor the coronary score differed among the ACE I/D genotypes （P〉0.05）. But the number of affected coronary vessels and the coronary score were significantly greater in the patients with the AT1R-AC genotype than in those with the AA genotype （P〈0.05）. In conclusion, DD genotype may be risk factor for CHD and MI in Chinese people, and is not responsible for the development of the coronary artery stenosis. The AT1R-C allele may increase the relative risk associated with the ACE-DD genotype, and may be involved in the development of the stenosis of coronary artery.

Cryptic species composition and genetic diversity within Bemisia tabaci complex in soybean in India revealed by mtCOI DNA sequence

Bemisia tabaci is a cryptic species complex, causing signiifcant loss on many agricultural y important crops worldwide. Knowledge on species composition and diversity within B. tabaci complex is critical for evolving sustainable pest management strategies. Here we investigate the whitelfy species complex in soybean in major soybean growing states of India. The mitochondrial cytochrome oxidase gene subunit-1 (mtCOI) based phylogenetic relationships established using Bayesian methods indicated the existence of three cryptic species namely Asia I, Asia II 1, and Asia II 7. Al the haplotypes detected in the study could be assigned to these three cryptic species fol owing the species demarcation criteria of 3.5%divergence threshold. Of these, Asia II 1 was found to be predominant with wide spread distribution across the surveyed regions from cool temperate zones to hot and humid tropical plains. On the contrary, cryptic species Asia II 7 showed localized distribu-tion. The Asia II 1 exhibited the highest haplotype diversity and Asia I showed high level of nucleotide diversity. There was a signiifcantly high genetic differentiation among these three cryptic species. The MEAM 1, a dreadful invasive species was not detected in the specimens tested in the current study. The diversity and distribution of three cryptic species is discussed in the light of current knowledge on distribution of whitelfy species in India and yel ow mosaic disease observed during sampling survey.

Micro Gene Sys公司开展疫苗试验

上周,用 MicroGenSys 公司(康涅狄格州)研制的 AIDS 疫苗在新的一类病人(即早期 HIV阳性个体)上进行了试验,这类病人感染后不产生发病征状。这项Ⅰ期临床研究正在这里的Walter Reed 军事医疗中心进行着。该中心情报官员 Paul Klein 说。

POLYMORPHISM OF ANGIOTENSIN I TYPE 1 RECEPTOR GENE IN ELDERLY PATIENTS WITH ESSENTIAL HYPERTENSION

Objective To detect the A/C1165 polymorphism of angiotensin Ⅱ type Ⅰ receptor (AT1-R)gene in essential hypertensive elderly. Methods The A/C1166 polymorphism of AT1-R gene was assessed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in a case-control study of 87 essential hypertensive elders (EH) and 55 normolensive elders (NT). Results The genotype frequencies of AA, AC, CC were 0 .805 , 0.161, 0 .034 in EH group and 0 .927 ,0 .073 ,0 .000 in NT group respectively. The frequency of C61166 allele was higher in EH group (0.115) than in NT group (0 .036 )(P<0 .05 ). Conclusion The resultsindicate that A/C1166 polymorphism of AT1-R gene may be associated with essential hypertension in elderly.

Between Scylla and Charybdis:The role of the human immune system in the pathogenesis of hepatitis C

Hepatitis C virus(HCV)frequently elicits only mild immune responses so that it can often establish chronic infection.In this case HCV antigens persist and continue to stimulate the immune system.Antigen persistence then leads to profound changes in the infected host’s immune responsiveness,and eventually contributes to the pathology of chronic hepatitis.This topic highlight summarizes changes associated with chronic hepatitis C concerning innate immunity(interferons,natural killer cells),adaptive immune responses(immunoglobulins,T cells,and mechanisms of immune regulation(regulatory T cells).Our overview clarifies that a strong anti-HCV immune response is frequently associated with acute severe tissue damage.In chronic hepatitis C,however,the effector arms of the immune system either become refractory to activation or take over regulatory functions.Taken together these changes in immunity may lead to persistent liver damage and cirrhosis.Consequently,effector arms of the immune system will not only be considered with respect to antiviral defence but also as pivotal mechanisms of inflammation,necrosis and progression to cirrhosis.Thus,avoiding Scylla-a strong,sustained antiviral immune response with inital tissue damage-takes the infected host to virus-triggered immunopathology,which ultimately leads to cirrhosis and liver cancerthe realm of Charybdis.

lacⅠ 靶基因突变子的一种正向选择系统的建立

目的：建立一种能正向选择ｌａｃⅠ靶基因突变子的选择系统。方法：比较了分别携带有ｌａｃⅠ－ ／ｌａｃＺα型质粒和ｌａｃⅠ＋ ／ｌａｃＺα型质粒的ＤＨ１０Ｂ菌在ＬＢ完全培养基、以葡萄糖为碳源的Ｍ９ 基本培养基和以乳糖为碳源的Ｍ９／Ｌ基本培养基内的生长情况。结果：组成型ＤＨ１０Ｂ菌在Ｍ９／Ｌ固体培养基中只需培养２３ ｈ 便可观察到生长，而诱导型ＤＨ１０Ｂ菌则需培养８８ ｈ 才可观察到生长，但在ＬＢ和Ｍ９ 培养基内两者的生长速度却相同；当将少量的组成型ＤＨ１０Ｂ菌与大量的诱导型ＤＨ１０Ｂ菌混合接种于Ｍ９／Ｌ固体培养基上培养时， Ｍ９／Ｌ固体培养基能从约２．７×１０６ 的ｌａｃⅠ＋ 型ＤＨ１０Ｂ菌中将少量的ｌａｃⅠ－ 型ＤＨ１０Ｂ菌筛选出来。结论：本研究建立了一种在特定时间内只选择生长携带有ｌａｃⅠ－ ／ｌａｃＺα型质粒的ＤＨ１０Ｂ菌落。

pM CLacⅠ/Neo质粒的构建和基因突变研究的新策略

目的：构建一个用于比较研究哺乳动物细胞内表达基因和沉默基因的不同突变机制的质粒载体。方法和结果：设计并构建一种具两个ｌａｃⅠ突变靶基因的突变研究载体：ｐ Ｍ Ｃ ＬａｃⅠ／ Ｎｅｏ 质粒。在该质粒中，一个ｌａｃⅠ靶基因受 Ｃ Ｍ Ｖ 启动子的驱动而使其在人和哺乳动物细胞内能表达，而另一个ｌａｃⅠ靶基因则不能表达。将所获得的ｐ Ｍ Ｃ ＬａｃⅠ／ Ｎｅｏ 质粒经酶切鉴定后，再进行如下功能鉴定：一是分析两个ｌａｃⅠ靶基因在大肠杆菌细胞内的功能状态，结果显示这两个ｌａｃⅠ基因在 Ｄ Ｈ５α宿主菌内功能正常；二是将其导入 Ｎ Ｉ Ｈ３ Ｔ３ 细胞内，分析两个靶基因是否能模拟哺乳动物细胞内表达基因和沉默基因的功能状态。结果表明其中一个ｌａｃⅠ靶基因在 Ｎ Ｉ Ｈ３ Ｔ３ 细胞中处于转录表达状态。结论：本研究构建了一种新型的突变研究载体，位于该载体上的两个ｌａｃⅠ靶基因能模拟人和哺乳动物细胞内表达基因和沉默基因的功能状态。

Angiotensin Ⅰ-converting enzyme gene poly morphism in Chinese patientswith obstructive sleep apnea syndrome

Objective To investigate the relationship of an insertion/deletion (I/D) polymorphism of the angiotension converting enzyme (ACE) gene to obstructive sleep apnea syndrome (OSAS) patients and the control subjects.Methods Genomic DNA was extracted from blood samples and amplified by polymerase chain reaction (PCR). PCR primers flanked the polymorphic region in intro 16 of the ACE gene. Results OSAS patients had significantly higher frequencies of I/I genotype and insertion allele of the ACE gene as compared with the control subjects in Chinese population. The OSAS patients with I/I genotype had significantly longer apnea time, lower minimum SaO2 and greater AHI than the OSAS patients with I/D genotype. Conclusion These results indicate that the I/I genotype and I allele are a risk factor for OSAS in Chinese.

pMCLacⅠ/neo转基因小鼠靶基因突变检测的新策略

由于pMCLacⅠ neo转基因小鼠含有两种不同状态的LacⅠ靶基因 ,有别于以往所建立的只含有一种靶基因的系统。因此 ,建立一种能快速、有效地分析这两种靶基因的检测方法 ,成为该转基因小鼠建立后的又一新课题。通过适当方法将pMCLacⅠ neo中两种LacⅠ靶基因分开后 ,采用了新近建立的M9 L正向选择系统进行检测 ,并用已知的LacⅠ基因突变子作为阳性对照 ,验证该策略的可行性。实验结果提示 ,M9 L正向选择系统可应用于pMCLacⅠ neo转基因小鼠中两种LacⅠ靶基因的检测 ;实验中所建立的突变检测方法快速。

Comparative study of catheter-mediated gene transfer into heart

OBJECTIVE: To investigate the feasibility and features of 3 catheter-mediated approaches of gene transfer into heart, including direct myocardial injection (DMI), coronary artery perfusion (CAP), and intrapericardial cavity injection (ICI). METHODS: Fifteen dogs were used, and 0.3 ml (1 x 10(9) pfu) of an adenovirus (Adex1SR LacZ) was injected into the heart by 3 methods. The dogs were killed 5 days following injection, and gene expressions in heart and liver were evaluated by histochemical analysis. RESULTS: The results showed that (1) the CAP method was relatively less damaging and induced sparse LacZ expression in the myocardium, and the gene expression was also found in both vessels within the myocardium and liver; (2) gene transfer by DMI resulted in intense LacZ expression around the injection accompanied by a local inflammatory response; (3) LacZ expression elicited by ICI was detected in either the inner surface of the parietal pericardium or epicardial surface of the heart, and also in the myocardium underlying the visceral pericardium. CONCLUSION: Three catheter-mediated methods of gene transfer into the heart may be used and a reasonable approach should be chosen according to purpose.

单纯疱疹病毒Ⅰ型载体中介lacZ基因导入猴嗅中枢皮质及其表达的研究

目的：研究用ＨＳＶ－１载体经猴嗅神经通路将外源基因导入中枢神经系统的方法。方法：将ＨＳＶ－１载体接种于猴嗅神经末梢，在不同时间取各部位脑组织检测ｌａｃＺ基因表达产物β－半乳糖苷酶活性，并观察猴脑组织局部及全身的病理学和免疫学变化。结果：接种ＨＳＶ－１载体后４ｄ在嗅中枢皮质神经元内检测到β－半乳糖苷酶活性，并持续１个月以上；脑组织及全身脏器未见病理学及免疫学改变。结论：ＨＳＶ－１载体可通过嗅神经通路将外源基因导入中枢神经系统并在其中表达，