Identification of Bupleurum Seeds by ITS2 Genetic Barcoding and Seed Morphology / 中国实验方剂学杂志

Objective:To accurately identify Bupleurum seeds by traditional morphological identification method combined with DNA barcoding technique. Method:A total of 41 seed samples on the market were collected and 75 ribosomal DNA internal transcribed spacer 2 (ITS2) sequences of 15 varieties were downloaded from the GenBank database as experimental materials. The seeds were measured and observed by stereomicroscope and vernier caliper, and their 1 000-grain weights were calculated. Genomic DNA was extracted from the seeds and used as a template, and ITS2 sequences were amplified using polymerase chain reaction (PCR) and bidirectional sequencing. Species identification was conducted based on BLAST method, neighbor-joining (NJ) phylogenetic tree method, Kimura two-parameter model (K2P) genetic distance method, and secondary structure of ITS2 sequence. Result:There were slight differences in the length, width, cross-section, and 1 000-grain weight among Bupleurum seeds from different origins. The ITS2 sequences of B. chinense seeds had 2 intraspecific variable sites and 3 haplotypes, the maximum intraspecific genetic distance (0.009) was far smaller than the minimum interspecific genetic distance (0.032). B. chinense and B. scorzonerifolium in the NJ phylogenetic tree were clustered into independent branches with good monophyletic property. The secondary structure of ITS2 sequences could make up for the shortcomings of NJ tree in identifying variants. The collected 41 seeds included 30 B. chinense seeds, 3 B. scorzonerifolium seeds, 5 B. falcatum seeds, 2 B. marginatum var. stenophyllum seeds, and 1 B. smithii var. parvifolium seeds. Conclusion:The B. chinense seeds on the market have problems of diverse sources and chaotic origins. Based on the combination of ITS2 gentic barcoding and seed morphological identification, the Bupleurum seeds can be accurately identified, which provides scientific bases for establishing the quality standard of Bupleurum seeds, standardizing the cultivation of B. chinense, and solving the quality problems of B. chinense from the source, and provides a reference for the accurate identification of other medicinal plant seeds or seed medicinal materials.

Advances in Bioculture Technology of Bupleuri Radix and Biosynthesis Pathways of Saikosaponins / 中国实验方剂学杂志

Synthetic biology is an emerging discipline that analyzes the biosynthesis pathways of active constituents in traditional Chinese medicine and explores genes involved in biosynthesis. Bupleuri Radix is one of the most commonly used Chinese medicinal materials with remarkable medicinal value, its index component is saikosaponins, which has significant anti-inflammatory, anti-viral and anti-tumor activities. However, the current wild resources of Bupleuri Radix have been destroyed, and there were some problems in the process of artificial cultivation. The application of biological culture technology and synthetic biology can expand the sources of saikosaponins and protect resources of Bupleuri Radix. The culture conditions of different plants can be followed without a fixed pattern, and the biosynthetic pathways of different medicinal active ingredients are also inconsistent. At present, there is no review report on the culture technology of Bupleuri Radix and the research on the biosynthesis pathway of saikosaponins. This paper introduces the research progress of biological culture techniques, such as callus culture, adventitious root culture, hairy root culture and suspension cell culture used in synthetic biology, and the biosynthesis pathway of saikosaponins and its key enzyme functional genes. It is suggested to optimize the biological culture technology of Bupleuri Radix by referring to the tissue culture technology of other traditional root medicinal materials, so as to provide a reference for the in-depth study on the biosynthesis pathway and metabolic regulation of saikosaponins.

Study on correlation between trace elements and active ingredient in glycyrrhizae radix et rhizome / 中国中药杂志

To clear the kinds of trace elements which is closely related with the active ingredient, proclaim the effects of trace elements on the quality of the Glycyrrhizae Radix et Rhizoma, provide the theoretical foundation to the further quality control of cultivation, take the advantage of the HPLC to determine the contents of glycyrrhizic acid and the liquiritin according to Chinese pharmacopoeia, use the inductively coupled plasma mass spectrometry to test the contents of Cu, Zn, Mn, Pb, Se, Cd, Ni, La, Na, Cr, M, Fe, Ca, Al, K, Sr, then, use SPSS statistical software for active ingredient and trace elements Correlation Analysis. The result of correlation analysis showed that Liquiritin contents of Glycyrrhizae Radix et Rhizoma have strong a positive correlation with the Mn, Pb contents, well, have a negative correlation with the Cu, Na contents; Glycyrrhizic acid contents showed a positive correlation with Mg, Cd, La contents, however, it showed a negative correlation with K, Fe contents. Comprehensive analysis of the results of the study, a preliminary thought that the active ingredient of Glycyrrhizae Radix et Rhizoma closely related with the trace elements, but the exact conclusion still need further study concentration-response relationship analysis.

Research advances of genomic GYP coding MNS blood group antigens / 中国实验血液学杂志

The MNS blood group system includes more than 40 antigens, and the M, N, S and s antigens are the most significant ones in the system. The antigenic determinants of M and N antigens lie on the top of GPA on the surface of red blood cells, while the antigenic determinants of S and s antigens lie on the top of GPB on the surface of red blood cells. The GYPA gene coding GPA and the GYPB gene coding GPB locate at the longarm of chromosome 4 and display 95% homologus sequence, meanwhile both genes locate closely to GYPE gene that did not express product. These three genes formed "GYPA-GYPB-GYPE" structure called GYP genome. This review focuses on the molecular basis of genomic GYP and the variety of GYP genome in the expression of diversity MNS blood group antigens. The molecular basis of Miltenberger hybrid glycophorin polymorphism is specifically expounded.

Application of microfluidic chip analytical systems in ABO genotyping / 中国实验血液学杂志

Limitations of polyacrylamide gel or agarose gel electrophoretic methods in genotyping research affect the interpreting of detection results. In order to develop a simple and reliable method for appraising results of ABO genotyping detection, the microfluidic chip analysis system was established by using microfluidic chip to replace the gel electrophoresis and combining with multiplex-PCR-RFLP technique. 150 blood samples were tested by this microfluidic chip analysis system with multiplex-PCR-RFLP technique to evaluate its stability and accuracy. The results showed that all the testing results were consistent with serologic ABO genotyping results and 1 blood sample with decrease of B antigen caused by CML was identified. In conclusion, the established microfluidic chip analysis system is stable and reliable technique. Application of this technique enables the ABO genotyping results to be more objective and accurate.

Study on geographical variation of morphologic and germination characteristic of different Glycyrrhiza uralensis provenance seeds / 中国中药杂志

<p><b>OBJECTIVE</b>To study the geographical variation of morphologic and germination characteristic of different Glycyrrhiza uralensis provenance seeds, approach the geographical variation mode and ecology mechanism, and laid theoretical foundation for districting and allocating of G. uralensis seeds.</p><p><b>METHOD</b>Field investigation and laboratory analysis were applied. Seed shape and kilosseed weight were sampled randomly, germination rate germination force by general methods.</p><p><b>RESULT</b>The morphologic characteristic of G. uralensis seeds showed roughly longitude variation tendency that the seeds increased gradually from west to east. While the germination characteristic showed roughly altitude variation tendency that the seeds germination rate and germination force increased with the increase of the altitude, and the average germination rate was the same with the seeds morphologic characteristic. The results of analysis correlated with the climatic factors show that the morphologic characteristic of G. uralensis was positive correlated with annual rain-fall of the habitat, and the germination rate was quickened by drought, high temperature and strong sunshine.</p><p><b>CONCLUSION</b>The morphologic and germination characteristic and of G. uralensis seeds present distinguished geographical variation, and the formation of the variation was related to the ecological environment in which the seed provenance adapted.</p>

ABO genotyping of Han population in Beijing / 中国实验血液学杂志

The aim of this study was to establish a diagnostic method for ABO genotyping and to investigate the distribution of ABO genotype in Beijing Han population so as to understand the distribution characteristics and regularity of ABO genotype. An ABO genotyping method was established by using multiplex-PCR-RFLP and PCR-SSP techniques, and the ABO allele frequency in Beijing Han population was investigated. The results showed that A102, O1 and B allele were more common genes in Beijing Han individuals. And A102 allele was predominant in the phenotype A group in this population. Three O2 alleles were found and no A201 allele was found while gene frequency investigation was performed. No A101A101, A101O2, A102O2, BO2 and O2O2 in this population were discovered. It is concluded that the primary regularity of ABO allele distribution in Beijing Han population is found through this study. It provides basic reference for further study of ABO types.

Stability of hepatitis C virus RNA in various processing and storage conditions / 中国实验血液学杂志

The study was purposed to investigate whether processing and storage conditions might influence the stability of the HCV RNA in whole blood or in plasma. The samples obtained from seven patients known to be positive for HCV RNA were kept in different storage conditions with different anticoagulants, and at the end of processing the plasma samples were frozen at -80 degrees C until fluorescent quantitative PCR testing. The results showed that there was no significant loss of HCV RNA titers in whole blood anticoagulated with CPDA or ACD or EDTA or none (P > 0.05), while differences in comparison of the EDTA-anticoagulant storage condition with three other anticoagulants storage conditions at 4 degrees C after 48 hours were significant (P < 0.05). The HCV RNA level decreased to 53.8%, 72.5% and 29.8% after 48 hours of storage of whole blood anticoagulated with ACD at 4 degrees C, 25 degrees C and 37 degrees C respectively. The HCV RNA level of plasma samples stored at 4 degrees C and at 25 degrees C (room temperature) after 7 days decreased to 70.9% and 25.1% respectively. After four freeze-thaw cycles the HCV RNA level decreased 38.9% in plasma samples. It is concluded that the HCV RNA is stable relatively. The HCV RNA is resistant to degradation under routine laboratory handling and storage conditions or blood collection, transport and processing conditions. The influence of different anticoagulants on the stability of HCV RNA is different. Blood samples would better be stored at 4 degrees C after collection and plasma separated within 48 hours. And it is important for the stability of HCV RNA undergoing asepsis blood collection process. HCV RNA remains stable at 4 degrees C for at least 7 days or at room temperature for 3 days, allowing greater flexibility in samples collection and transport in transfusion practice nowadays. HCV RNA in plasma samples subject to up to three short-term freeze-thaw cycles is still stable.

Correlation between signal/cutoff ratios of anti-HCV enzyme immunoassay (EIA) and their true positivity in blood donors / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To evaluate the correlation between signal/cutoff (S/CO) ratios of anti-HCV EIA and their true positivity for determining the predictive value of S/CO ratios.</p><p><b>METHODS</b>One hundred and fifty-nine samples of blood from donors positive for anti-HCV at the initial screening were collected from Beijing, Guangzhou, Hangzhou, Kunming and Urumchi. All the samples were retested by Ortho and 6 Chinese domestic anti-HCV EIA kits in duplicate, and detected for HCV RNA (NAT) using Chiron Procleix HIV/HCV system (transcription mediated amplification, TMA). The HCV RNA negative samples were further tested for anti-HCV by Chiron RIBA 3.0. Either NAT or RIBA positive samples were interpreted as the true positive.</p><p><b>RESULTS</b>All 7 anti-HCV EIA kits had a significant correlation between S/CO ratios and true positivity. The S/CO ratio of Ortho > or = 3.8 predicted the true positivity in 96.1% of the samples tested. The S/CO ratios of BGI-GBI, GWK, SABC, KHB, InTec, and Wantai were > or = 7.0, > or = 10.0, > or = 6.0, > or = 10.0, > or = 8.6, > or = 14.0 and predicted 96.1%, 96.1%, 97.3%, 96.0%, 96.1%, 96.0% of the true positivity, respectively.</p><p><b>CONCLUSIONS</b>The S/CO ratios of anti-HCV EIA kits are associated with the true positivity. S/CO ratios of Ortho, BGI-GBI, GWK, SABC, KHB, InTec and Wantai predicting > or = 95% true positivity are > or = 3.8, > or = 7.0, > or = 10.0, > or = 6.0, > or = 1 0.0, > or = 8.6 and > or = 14.0, respectively.</p>

Effect of drought stress on growth of Glycyrrhiza uralensis / 中国中药杂志

<p><b>OBJECTIVE</b>To study the effect of drought stress on the growth of Glycyrrhiza uralensis and its drought resistance characteristic.</p><p><b>METHOD</b>The growth in dicators of G. uralensis including leaf, stem, root and biomass were measured when two-years-old G. uralensis had lived in drought stress soil for 60 days.</p><p><b>RESULT</b>The growth of all organs of G. uralensis was restrained because of drought stress, especially to up ground organs.</p><p><b>CONCLUSION</b>It is obvious that the restraining effect of drought stress on G. uralensis possesses organ speciality. Both yield and quality of G. uralensis will be satisfied when it grows in the soil with relative water content of 50%.</p>

The associations of HLA-DQB1 gene with onset age and autoantibodies in type 1 diabetes / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To evaluate the associations of human leukocyte antigen (HLA) DQB1 gene with onset age and autoantibodies in type 1 diabetes mellitus(T1DM) in Chinese Han population in Sichuan area.</p><p><b>METHODS</b>Forty-six type 1 diabetic patients and 52 healthy control subjects were involved in this study. HLA-DQB1 typing was performed by polymerase chain reaction-sequence specific primer(PCR-SSP). Glutamic acid decarboxylase antibody (GADA) and islet cell antibody (ICA) were qualitatively analyzed by enzyme linked immunosorbent assay (ELISA).</p><p><b>RESULTS</b>The positive rate of DQB1*0201 was higher in T1DM than in controls (OR=18, P<0.005), but those of DQB1*0601, *0602 were higher in controls than in T1DM(OR=0.07, 0.31 respectively, both P<0.05).The positive rate of DQB1*0602 in type 1 diabetic patients with onset age>or=20 years was higher than that in the patients with onset age <20 years (P<0.05). GADA was more frequent in DQB1*0201(+) patients than in DQB1*0201 (-) patients (P<0.025).</p><p><b>CONCLUSION</b>The findings show that DQB1*0201 is susceptible to T1DM, whereas DQB1*0601, *0602 are protective in Chinese Han population in Sichuan area. DQB1*0602 may delay the onset of T1DM. The positive rate of DQB1*0201 correlates positively with that of GADA.</p>

The underground part growth distribution pattern of Glycyrrhiza uralensis and its effects on glycyrrhizinic acid content / 中国中药杂志

<p><b>OBJECTIVE</b>To ascertain the relationship between glycyrrhizinic acid content and the underground part growth character of Glycyrrhiza uralensis and provide the theoretical evidence for wild resources protection and artificial cultivation method of G. uralensis.</p><p><b>METHOD</b>Through the analytical investigation on the underground part of G. uralensis and analysis of glycyrrhizinic acid content in different organs, parts, ages, and diameter medicinal materials, the systematic study on the relationship between glycyrrhizinic acid content and the underground part growth character of glycyrrhiza uralensis was carried out.</p><p><b>RESULT</b>The underground part of a G. uralensis seedling consisted of seed root, random root, horizontal underground stem, vertical underground stem and assimilating root. The glycyrrhizinic acid content in horizontal underground stem with the age below two years old or in random root with the diameter below 0.5 cm was low. The difference of glycyrrhizinic acid content among horizontal underground stem, random root and vertical underground stem was obvious, but the difference between horizontal underground stem and random root was not obvious.</p><p><b>CONCLUSION</b>The horizontal underground stem was of G. uralnesis acts as a link that can connect random root, vertical underground and stem assimilating root, so that the whole underground part constructs one huge underground net system. The glycyrrhizinic acid accumulation is a effected by organ type, growth age, root diameter and grow position, and the distribution pattern of random root and vertical underground stem has influence on glycyrrhizinic acid distribution in horizontal underground stem.</p>

Establishment of a sandwich ELISA method for detection of matrix metalloproteinase 9 protein in sera samples and preliminary application for hepatocellular carcinoma patients / 中华检验医学杂志

Objective Expression of recombinant human matrix metalloproteinase 9 (MMP9) protein in E coli,To establish a sandwich ELISA method for detecting MMP9 in sera and analyses of the difference of serum MMP9 in health population and the patient with hepatocellular carcinoma (HCC). Methods A fragment MMP9 sequence from nt 721-1 156 was amplified with PCR from the synthesized cDNAs of human liver tissues and inserted into the prokaryotic-expression plasmid pQE30.HIS-MMP9 fusion protein was expressed and purified from E coli MIS cells.The specific antibodies were elicited in rabbits and guinea pigs by immunization of the purified HIS-MMP9.After purifing antibodies with CNBr-activated Sepharose 4B,a sandwich ELISA technique was established.The serum MMP9 proteins were evaluated in 227 health adults and 193 HCC patients.Results SDS-PAGE displayed that the molecular-weight of the expressed fusion protein was about 17 000.The prepared antisera were able to recognize both recombinant and endogenously expressed MMP9 from human neutrophil and HepG2 cells.It was found that the average level of MMP9 proteins in HCC patients was higher than that in health control,showing significantly statistic difference.Conclusions The established method could reflect the level of serum MMPg.The data in this study supply scientific basis of generating methodology for screening metastasis and reoccurrence of HCC, using serum MMP9 as index.