Studies on constituents of Polygonum multiflorum extract and based on UPLC-Q-TOF-MS / 中国中药杂志

To explore the drug-induced constituents of Polygonum multiflorum extract (PM). This study was the first to study the drug-induced constituents in target organ liver. Agilent MassHunter qualitative analysis software and Metabolite ID software were applied for the analysis of retention time, exact relative molecular mass, primary and secondary mass spectrum information based on ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) and targeted-MS/MS. By comparison with literature and standards, a total of 5 prototypes and 6 metabolites were identified or tentatively elucidated from the liver samples. In addition, the drug-induced constituents in plasma and PM were also analyzed in this study and 8 prototypes and 19 metabolites were detected from the plasma samples, while 30 compounds were detected from the extract of PM. Emodin oxidative acetylation metabolites, hydroxyl methylation metabolites, carboxylation glucuronidation metabolites and ketone glucuronidation metabolites in this study were first reported. Through the comparative analysis between the and constituents of PM, the study preliminarily revealed the drug-induced constituents (prototypes and metabolites) in liver and clarified the transfer process and transmutation rules of constituents in PM, blood and liver, which would further deepen our understanding on constituents of PM .

Effect of Zhuangyao Jianshen Wan (ZYJCW) on P2X1 and P2X3 mRNA expressions in rats with diuresis caused by kidney deficiency / 中国中药杂志

To investigate the urination-reducing effect and mechanism of Zhuangyao Jianshen Wan (ZYJCW). In this study, SI rats were subcutaneously injected with 150 mg · kg(-1) dose of D-galactose to prepare the sub-acute aging model and randomly divided into the model group, the Suoquan Wan group (1.17 g · kg(-1) · d(-1)), and ZYJCW high, medium and low dose groups (2.39, 1.20, 0.60 g · kg(-1) · d(-1)) , with normal rats in the blank group. They were continuously administered with drugs for eight weeks. The metabolic cage method was adopted to measure the 24 h urine volume and 5 h water load urine volume in rats. The automatic biochemistry analyzer was adopted to detect urine concentrations of Na+, Cl-, K+. The ELISA method was used to determine serum aldosterone (ALD) and antidiuretic hormone (ADH). The changes in P2X1 and P2X3 mRNA expressions in bladder tissues of rats were detected by RT-PCR. According to the results, both ZYJCW high and medium dose groups showed significant down-regulations in 24 h urine volume and 5 h water load urine volume in (P <0.05, P <0.01), declines in Na+ and Cl- concentrations in urine (P <0.01), notable rises in plasma ALD and ADH contents (P <0.05, P <0.01) and remarkable down-regulations in the P2X1 and P2X3 mRNA expressions in bladder tissues (P <0.01). The ZYJCW low dose group revealed obvious reductions in Na+ and Cl- concentrations in urine (P <0.01). The results indicated that ZYJCW may show the urination-reducing effect by down-regulating the P2X1 and P2X3 mRNA expressions in bladder tissues of rats with diuresis caused by kidney deficiency.

Construction and application of a multiplex PCR system for genotyping of human red blood cell antigens / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>A reliable method for genotyping blood group antigens Dib, k, Jsb1910 and Jsb2019 was developed. Through screening for rare blood types, the National Rare Blood Bank of China may be enriched.</p><p><b>METHODS</b>The controls for allele detection of blood groups Dib, k, Jsb1910 and Jsb2019 were prepared via polymerase chain reaction (PCR)-mediated gene site-directed mutagenesis (SDM) technique. Sequence-specific primers were designed according to known single nucleotide polymorphism (SNP) sites of alleles of blood groups antigens Dib, k, Jsb1910 and Jsb2019, a multiplex PCR system was developed by optimizing PCR reaction system. And 4190 random healthy donors samples were screened for the blood group antigens.</p><p><b>RESULTS</b>Using SDM technique, controls for alleles in blood group Dib, k, Jsb1910 and Jsb2019 were successfully generated. And a multiplex PCR system for genotyping above blood groups was developed. After verification, the system has performed with good stability and reproducibility. Two Di (b-) samples have been discovered from 4190 samples, no k- and Js(b-) sample was found.</p><p><b>CONCLUSION</b>Multiplex PCR features rapid detection, high throughput and low cost, and can be used for screening for donors of rare blood types. Information of donors may be registered in a database, which in turn can help those with rare blood types or require long-term blood transfusion to obtain matched blood, thereby reduce the adverse reactions of blood transfusion.</p>

The establishment of the controls for blood group genotyping and the application in the screening of three rare blood groups / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish the controls for allele detection of blood groups s and Ok(a). A multiplex PCR method for the detection of three blood group antigens Fy(a), s and Ok(a) was developed and used to investigate the distribution of these blood groups in Chinese random blood donors.</p><p><b>METHODS</b>Polymerase chain reaction (PCR)-based, gene site-directed mutagenesis (SDM) technique were used to make site-directed mutagenesis for the single nucleotide polymorphism (SNP) sites of the blood group alleles (the 153 C/T point mutation of the GYPB gene, and the 274 G/A point mutation of the BSG gene) as controls for allele detection. Sequence specific primers were designed according to the SNP sites of alleles of blood group antigens Fy(a), s and Ok(a). A multiplex PCR system was developed and 438 random donor samples were screened for the blood group antigens Fy(a), s and Ok(a).</p><p><b>RESULTS</b>The controls for alleles in blood groups s and Ok(a) were successfully made with the SDM technique, a multiplex PCR system was set up and successfully used to analyze the genotypes of three blood group antigens Fy(a), s and Ok(a). Two Fy(a-) samples were detected in the 438 samples, no s- and Ok(a-) sample was found.</p><p><b>CONCLUSION</b>The PCR-based SDM technique can be used to obtain the unavailable controls in blood group genotyping. The multiplex PCR technique established in this study is an efficient genotyping method for blood groups Fy(a), s and Ok(a).</p>

Analysis of Kell blood group system using polymerase chain reaction-restriction fragment-single strand conformation polymorphism combined with heteroduplex in Chinese / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the polymorphism of Kell blood group system in Chinese and to find a suitable method for large scale screening.</p><p><b>METHODS</b>An analysis method of polymerase chain reaction-restriction fragment-single strand conformation polymorphism (PCR-RF-SSCP) combined with heteroduplex was established to detect abnormal sample in KEL exon 7-9 area, then sequencing was used to find out the mutation site.</p><p><b>RESULTS</b>Two mutations were found from 500 samples: 966G > A mutation in exon 9 and C > A mutation in 67th site of intron 7, both with no amino acid change. The mutation rate was 4/1000. No mutation was found as missed in using PCR-RF-SSCP combined with heteroduplex.</p><p><b>CONCLUSION</b>PCR-RF-SSCP combined with heteroduplex is confirmed as an effective, economical and simple method, it is quite suitable for large scale population screening study with unclear gene background and unavailable positive controls. Since there is special polymorphism for Kell blood group system in Chinese, further study is needed.</p>

Study on the molecular background of Del phenotype in Chinese population / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To elucidate the molecular background of Del phenotype in the Chinese population and explore new Del alleles.</p><p><b>METHODS</b>Five hundred and fifteen RhD negative blood samples was tested by Rh typing test, indirect antiglobulin test and adsorption and elution assay to screen the Del phenotype. DNA of all the Del samples was analysed by multiplex polymerase chain reaction (MPX PCR) for the presence of RHD and by sequence-specific primer polymerase chain reaction (PCR-SSP) for Del alleles: RHD 1227A and RHD 885T. Samples which showed the negative result by PCR-SSP, were additionally analysed by genomic DNA sequencing and cDNA sequencing.</p><p><b>RESULTS</b>Seventy-nine Del samples were found by adsorption and elution assay. All these samples had RHD exons 3, 4, 5, 6, 7 and 9. Except 4 Del samples, other 75 Del samples carried the RHD 1227A allele. None of the samples had the RHD 885T allele. Four novel RHD alleles were found in these four Del sample. There were RHD 3G-->A (GenBank DQ310735), RHD 28C-->T, RHD 53T-->C (GenBank DQ451877,DQ451878), RHD 251T-->C (GenBank DQ310734).</p><p><b>CONCLUSION</b>fnRh blood group system is very complex. New D variation phenotypes and new RHD alleles may be discovered ceaselessly.</p>

Molecular study on CisAB and B(A) blood group in Chinese individuals / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>This is a study on some ABO subgroup samples which show discordant results of serological and molecular blood typing, the aim is to clarify their true ABO type by means of nucleotide analysis on exons 6 and 7 of their ABO gene.</p><p><b>METHODS</b>Absorb-elution test and family investigation were conducted to study 7 samples which were involved in ABO grouping discrepancies. Duplex polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) method was used to identify their ABO genotypes. PCR products of exons 6 and 7 were cloned and sequenced.</p><p><b>RESULTS</b>All the 7 ABO subgroup samples with the discordant results of serological and molecular blood typing were found to have the normal O gene. Four out of them were typed as ABsub by serology, they were all of the A*102/O genotype. Sequencing analysis found all their A gene having the nt467 (C-->T) and nt803 (G-->C) mutation by comparison with the A*101 allele, i.e. their real type should be CisAB/O. Three out of 7 were typed as AsubB by serology and as BO by genotype; and point mutation was detected in all of their B gene. One of them had the nt700 (C-->G) mutation, the other 2 unrelated individuals had the novel nt640 (A-->G) mutation in their B alleles.</p><p><b>CONCLUSION</b>Through nucleotide analysis, 7 samples have been typed as AB subgroup in serology with the normal O gene, their real ABO type being CisAB in 4 cases and B(A) in 3 cases. At the same time, a kind of novel B (A)640 allele has been uncovered in this study.</p>

Analysis on FUT1 and FUT2 gene of 10 para-Bombay individuals in China / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>This is a study on the allele composing of ABO, FUT1 and FUT2 gene loci of 10 para-Bombay individuals in China.</p><p><b>METHODS</b>Ten samples coming from different districts of China were suspected of para-Bombay phenotype by primary serology tests. Routine and absorb-elution tests were conducted to identify their ABO type, and duplex polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was applied to getting their ABO genotype. Most of them were submitted to a test of their Lewis type as well. Then through direct DNA sequencing with PCR products of FUT1 and FUT2 genes, the genotypes of their H and SE gene loci were analyzed.</p><p><b>RESULTS</b>It can be confirmed that the 10 samples are para-Bombay. All of their ABO genotypes are consistent with the serological absorb-elution results and the substances detected results in saliva. Seven out of 10 have recessive homozygous gene at their H locus. Each phenotype of h1h1 (nt547-552Deltaag), h2h2 (nt880-882Deltatt) and h4h4 (nt35 t-->c) are ascertained in 2 individuals; moreover, h3h3 (nt 658 c-->t) is identified in one individual. The rest are hh heterozygous individuals: one is h3/h(new-1); the other is h2/h(new-2); the last one is h1/h2. The h(new-1) (nt586 c-->t) allele has a point mutation at nt 586 C to T, which leads a nonsense mutation Gln(CAG) to stop (TAG).The second h (new-2) (nt328 g-->a) has an nt328 G to A missense mutation,which leads Ala (GCC),was replaced by Thr (ACC) at 110 amino acid position. All the 10 samples have Se (nt357 c-->t) synonymous mutation. One Bm(h) (B/O) individual with h4h4 phenotype has a Se(w)(nt357 c-->t; nt385 a-->t) allele, whose Lewis type is Le(a+b+). Moreover, the authors detected a (nt716 g-->a) mutation in two samples' Se gene.</p><p><b>CONCLUSION</b>Four kinds of known h alleles (h1-h4), 2 kinds of novel non-functional FUT1 alleles, a Se(w) allele, and a novel SeG716A polymorphism in Chinese para-Bombay individuals were detected. At the same time, the authors noticed that all the 10 samples have the nt357 c-->t mutation in their FUT2 gene.</p>