ABSTRACT
Non-invasive prenatal testing (NIPT) has been extensively applied in fetal chromosomal aneuploidy screening with high sensitivity and specificity.The concentration of cell-free fetal DNA (cffDNA) is an important factor related to the accuracy of NIPT.When cffDNA concentration is lower than 4％,accurate results are often unavailable,resulting in failure of NIPT.The factors influencing cffDNA concentration in maternal blood were reviewed and it was found that the concentration of cffDNA was positively correlated with the gestational week at the time of sampling,but negatively correlated with the maternal weight/body mass index.Moreover,gestational complications (such as preeclampsia and polycystic ovary syndrome),maternal age,results of serological screening for Down's syndrome in second trimester,fetal chromosomal karyotype and nuchal translucency thickness were also potential influencing factors.Analysis of the influencing factors of cffDNA concentration helps to improve the NIPT specifications,which is significant for prenatal genetic counselling.
ABSTRACT
Objective To clone and express cathepsin B gene of Echinococcus granulosus(EgCatB)and analyze EgCatB protein by using bioinformatics tools and online databases. Methods The total RNA of E. granulosus was extracted and reverse?ly transcribed into cDNA as the template sequence for PCR. The EgCatB gene was cloned by using the In?Fusion PCR cloning method and expressed by a wheat germ cell?free system,and then the recombinant protein was identified by Western blotting. The signal peptide,transmembrane helices and subcellular location of the EgCatB sequence were predicted by the online soft?ware SignalP 4.1,TMHMM sever v. 2.0 and TargetP 1.1 respectively. Subsequently,the homologue sequence and conserved sites were aligned by using BLASTP and GeneDoc software. Finally,the structures and the glycosylation modification site of the EgCatB encoding protein were analyzed and predicted in turn by ProtParam,SMART,Predictprotein,Swiss?model,NetOGlyc 4.0 and NetNGlyc 1.0 approaches. Results The EgCatB gene was successfully amplified from cDNA of E. granulosus and ex?pressed in the soluble fractions. The molecular weight of the expressed protein was estimated 35 kDa. The bioinformatics analysis revealed that EgCatB was a classical secreted protein containing a Pept_C1 domain. The homology analysis indicated that the amino acid sequence of EgCatB was highly conserved in the active enzyme sites. The protein structure prediction showed a cata?lytic active center was formed through Gln106,Cys112,His282 and Asn302. It was found that there were nine O?glycosylation sites in the EgCatB sequence,but no N?glycosylation sites. Conclusions The EgCatB gene is cloned and expressed successfully,and the recombinant protein is analyzed by bioinformatics approaches and structure predication. The study provides useful informa? tion for further functional study of the EgCatB protein.
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Objective To study the inhibitory effect of pterin acid (PTA) against ricin and recombinant ricin A chain protein. Methods Luciferase protein synthesis inhibition assay in a cell-free system and in vitro cytotoxicity experiments were performed to assess the biological activity of ricin and rRTA treated with PTA.Results The result showed that PTA could significantly inhibit the activity of ricin and rRTA in a dose-dependent manner.Conclusion PTA might be used as a small molecular probe to develop an evaluating system for ricin/RTA small molecular inhibitor in vitro. The cell-free system adopted in the current study could also serve as a necessary basis for screening some novel small molecular compounds against ricin and RTA in the future.
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The purpose of this study was to examine reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, - a generated superoxide - of neutrophils in human peripheral blood after maximal exercise. Ten healthy male college students (20.2 ± 0.4 yr, 170.5 ± 1.3 cm, 62.8 ± 1.9 kg) participated after giving written informed consent. They performed an incremental exercise to volitional exhaustion using a bicycle ergometer. Peripheral blood was collected before exercise (Pre), just after exercise (Post) and 1-hour after exercise (Post-1h). Phorbol 12-myristate 13-acetate (PMA)-stimulated and opsonaized zymosan (OZ)-stimulated superoxide-generating activity of neutrophils was measured by the cytochrome c reduction assay. NADPH oxidase activity was measured by a cell-free system. NADPH oxidase activity significantly decreased in Post-1h compared with Pre and Post. A similar tendency was seen in PMA-stimulated activity, but not in OZ-stimulated activity. A strong positive relationship between NADPH oxidase activity and PMA-stimulated activity was found in Pre and this relationship attenuated after exercise. NADPH oxidase activity was not related to OZ-stimulated activity at any time points. We concluded that NADPH oxidase activity decreased after exhaustive maximal exercise in human peripheral neutrophils, and suggest that PMA-stimulated activity, relatively - speaking, reflects NADPH oxidase activity; but OZ-stimulated activity is independent of NADPH oxidase activity.
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Protein is one of major bio-functional performers. As one of several crucial proteomic research approaches, protein microarray has these following advantages: high-throughput, high sensitivity, quick detection and so on. Meanwhile, there are some critical factors that are important to the further development of protein microarray technology, for example, how to express and purify proteins for the research of protein microarray, how to immobilize proteins onto the substrate and keep the bio-function of proteins immobilized. Nano-biotechnology and cell-free expression system have been used to fabricate protein microarray by the way of immobilizing target genes onto the substrate and directly expressing corresponding proteins, which provides a new strategy to fabricate more complicated microarray. The stragegy and its progress were summarized———fabrication of protein microarray based on DNA, including immobilization of target genes, cell-free expression to proteins, immobilization of renascence proteins, advantages and drawbacks of the methods of protein chip fabrication etc.
ABSTRACT
Nuclear reconstitution around Lambda DNA in a cell-free system from Xenopus eggs involves distinct steps at ultrastructural level. First, Lambda DNA polymers were induced to form chromatin-like structures with the proteins in egg extracts. Then, along with membrane vesicles and nuclear pore components attached to them to assemble double nuclear membranes, these chromatin-like structures underwent variations from condensation to decondensation, simultaneously. It is different from the nuclear reconstitution induced by chromatin in that, while membrane vesicles were attaching to the chromatin-like structures to fuse each other, the assembly of nuclear pore complexes occurred practically.