自组装短肽GFS-4作为细胞三维培养及心肌梗死修复支架材料的研究EI北大核心CSCD

本研究探索自组装短肽GFS-4在心肌细胞三维培养中的应用效果及其对心肌梗死区域的组织修复作用。通过圆二色谱仪分析短肽GFS-4的二级结构,原子力显微镜检测短肽GFS-4自组装的微观形态。将GFS-4自组装形成的纳米纤维支架作为心肌细胞三维培养材料,观察心肌细胞的生长状况;建立大鼠心肌梗死模型,加入水凝胶GFS-4研究其对心肌梗死修复的效果。结果发现,GFS-4自组装形成的二级结构主要为β折叠;自组装24 h后形成致密的纳米纤维支架;心肌细胞三维培养结果表明心肌细胞在GFS-4水凝胶中生长状况良好;心肌梗死体外修复实验发现,短肽GFS-4水凝胶支架可缓解心肌梗死区域组织坏死。自组装短肽GFS-4作为新的纳米支架材料,可用于细胞三维培养和心肌梗死区域组织修复。     

新型手性自组装短肽R-LIFE-1的理化性质及其对外泌体的控释研究EI北大核心

本研究旨在探究手性自组装短肽R-LIFE-1对外泌体的包裹控释作用。采用原子力显微镜、透射电镜及冷冻扫描电镜等观察短肽R-LIFE-1的成胶能力及形态结构;采用光镜观察及荧光染色探究R-LIFE-1的生物相容性;采用超滤离心法提取外泌体并采用Western blot、纳米粒径追踪分析(NTA)、透射电镜检测外泌体质量;采用激光共聚焦显微镜及BCA蛋白定量探究短肽水凝胶对外泌体的控释效果。结果显示手性自组装短肽R-LIFE-1能够在离子触发下快速自组装形成稳定的纳米纤维网络膜片状结构,具有良好的生物相容性,能够负载外泌体对它进行包裹控释,提高外泌体的利用效率。本研究证明短肽R-LIFE-1可对外泌体进行控释,是一种理想的组织工程材料。     

自组装肽纳米纤维支架及其在组织工程的应用

自组装肽纳米纤维支架(SAPNS)是近年来被广泛研究的一种生物支架材料,其在三维细胞培养、组织修复与再生等领域得到了越来越多的应用。本综述在介绍SAPNS研究现状的基础上,按外、中、内3个胚层来源组织的顺序阐述了SAPNS在组织工程中的应用进展、存在问题及展望。     

手性自组装短肽Sciobio Ⅱ、Sciobio Ⅳ用于细胞三维培养和兔前交叉韧带损伤修复

目的 探究手性自组装短肽Sciobio Ⅱ、Sciobio Ⅳ对前交叉韧带损伤修复效果.方法 通过圆二色谱仪、透射电镜、苯胺蓝染色分析自组装短肽Sciobio Ⅱ、Sciobio Ⅳ的结构;利用吖啶橙/溴化乙锭(AO/EB)染色、异硫氰酸荧光素标记的鬼笔环肽染色检测三维环境中人韧带成纤维细胞(HLF)的活性和形态;建立...     

内皮素—1基因内含子4Taq I片段多态性的研究

根据EDN1第4内含子设计相应引物,建立检测EDN1第4内含子TaqI多态性PCR＋RFLP技术。利用该技术,扩增到具有特异性的PCR产物,长为358bp。经限制性核酸内切酶TaqI酶酶切检测中国汉族人群该片段多态性状况。实验显示中国汉人群中存在该位点的多态性。基因T1的频率为0．664;T2基因型频率为0．336;T1T1基因型频率为0．418;T1T2基因型频率为0．492;T2T2基因型频率为0．090。该位点可作为遗传标记探讨中国人群中与EDN1相关遗传病的关系。     

Improvement of HLA class I and class II PCR-SSP typing by using timed-release activity of DNA polymerase

Abstract: Variable amounts of non-specific amplification may occur in HLA PCR-SSP typing, and this can be significantly reduced by the use of AmpliTaq Gold. In an effort to achieve an optimal balance between specificity and efficiency of the PCR amplification for both HLA alleles and the internal control, we designed a system of timed-release activity by combining two different Taq DNA polymerases. The reaction was started at a relatively low level of enzyme activity and as thermal cycling progressed more and more Ampli-Taq Gold was slowly activated for the reaction to continue. We applied this system to all routine HLA PCR-SSP typing. The number of repeat typings due to non-specific amplification and/or amplification failure of the internal control was remarkably reduced.     

Identification of the yeast DNA polymerase I gene with antibody probes

Summary Partially overlapping fragments of the gene encoding yeast DNA polymerase I have been cloned by immunological screening of a yeast genomic library constructed in the phage expression vector gt11. The three gene fragments we analyzed in detail encode part of a yeast protein that has been identified as yeast DNA polymerase I, because it shares with this enzyme a number of antigenic determinants. In fact, the yeast protein fragments expressed by the recombinant phages react with both polyclonal and monoclonal antibodies raised against different, highly purified preparations of DNA polymerase I. Moreover, they can be used to affinity purify antibodies specifically reacting with active DNA polymerase I polypeptides and they compete with the yeast enzyme for binding to antibodies that inhibit catalytic activity. The gene is located on chromosome XIV in the yeast genome, and it is transcribed as a 5.2 kb mRNA.     

Functional characterization of partially purified Epstein-Barr virus DNA polymerase expressed in the baculovirus system

The DNA polymerase gene of Epstein-Barr virus (EBV) was cloned into baculovirus transfer vector (pBlueBac). The recombinant baculovirus (AcEBP-15) was obtained by cotransfection ofSpodoptera frugiperda (Sf9) cells with infectious DNA fromAutographa californica multiple nuclear polyhedrin virus (AcMNPV) and pBlueBac plasmid carrying EBV polymerase gene. Infection of Sf9 cells with the recombinant virus produced substantial quantities of the EBV DNA polymerase protein of the expected size (110 kD). The identity of the EBV polymerase 110-kD polypeptide was determined by (a) immunoprecipitation and Western blot analyses with rabbit polyclonal antiserum specific for a synthetic peptide derived from the coding sequence of the polymerase gene; (b) identification of a polypeptide of identical size (110 kD) from EBV-infected cells; (c) measurement of DNA polymerase activity similar to that of the enzyme induced in EBV-infected cells; and (d) neutralization of the enzymatic activity by the rabbit antiserum and inhibition by phosphonoacetic acid. Our results indicate that the baculovirus expression system provides large quantities of functional polymerase suitable for biochemical and structural analyses, thereby furthering our understanding of the mechanism of viral DNA replication and its inhibition by antiviral drugs.     

Rapid preparation of diagnostic probes for the fragile X syndrome by direct PCR amplification of human chromosomal DNA

Summary The fragile X syndrome is a common familial form of mental retardation and is associated with a rare fragile site at Xq27.3 (FRAXA). This disorder has recently been reported to correlate with length variations of restriction genomic DNA fragments which may due to the amplification of (CCG)_n trinucleotide repeats located at the FRAXA locus. We described here a rapid preparation method of diagnostic DNA probes for the fragile X syndrome by direct enzymatic amplification of human chromosomal DNA. ThePstI-assay, which is Southern blot analysis of DNA samples probed by PCR products, was shown to be sensitive method for diagnostic purposes to detect the size variations specific in the fragile X syndrome.     

Strategy for identifying the gene encoding the DNA polymerase of molluscum contagiosum virus type 1

Molluscum contagiosum virus (MCV) is a member of the family Poxviridae and pathogenic to humans. MCV causes benign epidermal tumors mainly in children and young adults and is a common pathogen in immunecompromised individuals. The viral DNA polymerase is the essential enzyme involved in the replication of the genome of DNA viruses. The identification and characterization of the gene encoding the DNA polymerase of molluscum contagiosum virus type 1 (MCV-1) was carried out by PCR technology and nucleotide sequence analysis. Computer-aided analysis of known amino acid sequences of DNA polymerases from two members of the poxvirus family revealed a high amino acid sequence homology of about 49.7% as detected between the DNA polymerases of vaccinia virus (genus Orthopoxvirus) and fowlpoxvirus (genus Avipoxvirus). Specific oligonucleotide primers were designed and synthesized according to the distinct conserved regions of amino acid sequences of the DNA polymerases in which the codon usage of the MCV-1 genome was considered. Using this technology a 228 bp DNA fragment was amplified and used as hybridization probe for identifying the corresponding gene of the MCV-1 genome. It was found that the PCR product was able to hybridize to theBamHI MCV-1 DNA fragment G (9.2 kbp, 0.284 to 0.332 map units). The nucleotide sequence of this particular region of the MCV-1 genome (7267 bp) between map coordinates 0.284 and 0.315 was determined. The analysis of the DNA sequences revealed the presence of 22 open reading frames (ORFs-1 to-22). ORF-13 (3012 bp; nucleotide positions 6624 to 3612) codes for a putative protein of a predicted size of 115 kDa (1004 aa) which shows 40.1% identity and 35% similarity to the amino acid sequences of the DNA polymerases of vaccinia, variola, and fowlpoxvirus. In addition significant homologies (30% to 55%) were found between the amino acid sequences of the ORFs 3,-5,-9, and-14 and the amino acid sequences of the E6R, E8R, E10R, and a 7.3 kDa protein of vaccinia and variola virus, respectively. Comparative analysis of the genomic positions of the loci of the detected viral genes including the DNA polymerases of MCV-1, vaccinia, and variola virus revealed a similar gene organization and arrangement.     

Role of DNA polymerase beta in the genotoxicity of arsenic

Arsenic, an important hazard in the environment, is associated with human cancer and other degenerative diseases. However, the mechanisms underlying arsenic hazardous effects remain unclear. It has been reported arsenic exposure can result in increased cellular reactive oxygen species and oxidative DNA damage. This suggests DNA base excision repair (BER), the major pathway for repairing oxidative DNA damage, may be involved in combating arsenic hazardous effects. As a critical repair enzyme in BER, DNA polymerase beta (Pol β) might play an essential role in reducing arsenic toxicity. To test this hypothesis, we evaluated arsenic-induced cytotoxic and genotoxic effects under Pol β deficiency. Our results demonstrated that the viability of Pol β-deficient mouse embryonic fibroblasts was much lower than that of Pol β wild-type cells after treatment with arsenite (As(3+) ). An increased level of DNA damage and significantly delayed arsenite-induced DNA damage repair in Pol β-deficient cells indicated reduced repair of DNA lesions under Pol β deficiency. This was consistent with the increase in the frequency of micronuclei (MN), an indicator of chromosomal breakage, which was also observed in Pol β-deficient cells treated with arsenite. In contrast, cells harboring overexpressed Pol β resulted in a lower level of DNA damage and MN than Pol β wild-type cells, indicating overexpression of the enzyme can combat arsenic-induced genotoxic effects. In conclusion, our results indicate an important role for Pol β in repairing arsenite-induced DNA damage and maintaining chromosomal integrity and further suggest deficiency of BER may be involved in arsenic genotoxicity and carcinogenicity.     

DNA polymerase alpha defect in the N syndrome

The segregation of genetic hemochromatosis was analyzed by using percent transferrin saturation (TS) as a phenotypic marker of the disease. Homozygotes for the disease were readily discernable with the added information provided by the quantitative indicator. However, there was no evidence of partial expression of TS abnormalities in heterozygotes, contrary to previous studies.