This study was aimed to establish the reliable genotyping method of human platelet antigens of HPA-15 system by PCR-SSP and to use this assay in the further HPA genotyping of volunteer platelet donors. 3 sequence-specific primers recommended by the 11th Platelet Genotyping and Serology Workshop on behalf of International Society of Blood Transfusion (ISBT) were synthesized. The concentration of each primer pair, the concentration of Mg(2+) and the PCR conditions were adjusted to optimize the conditions so that HPA-15 system could be specific amplified. The accuracy and reliability of the developed assay was evaluated and confirmed by typing the coded DNA samples provided by the 11th Platelet Genotyping and Serology Workshop. As a parallel control, a total of 50 volunteer platelet donors in Shenzhen were genotyped by both our assay and the G&T commercial kit at HPA-15 system. 10 coded samples distributed by the 11th Platelet Genotyping and Serology Workshop were genotyped by established PCR-SSP method. The results showed that a concordance rate of 100% was observed between the results obtained by established PCR-SSP method and the results provided by ISBT report. The HPA gene frequencies observed in 50 randomly-selected platelet donors in Shenzhen were 0.5100 and 0.4900 for HPA-15a and HPA-15b respectively. In conclusion, PCR-SSP assay established in our study provides a simple, rapid and accurate method for HPA-15 system genotyping, which assay is suitable for routine clinical HPA genotyping and shows a broad prospect in its further applications.
Antigens, CD ; genetics ; immunology ; Antigens, Human Platelet ; genetics ; GPI-Linked Proteins ; Genotype ; Humans ; Isoantigens ; genetics ; immunology ; Neoplasm Proteins ; genetics ; immunology ; Polymerase Chain Reaction ; methods ; Polymorphism, Single-Stranded Conformational

OBJECTIVETo develop a reliable assay for simultaneous sequence-based typing (SBT) of HLA-DPA1 and HLA-DPB1, and to apply it for the study of allelic polymorphisms in southern Chinese Han population.

METHODSBased on full-length HLA-DPA1 and HLA-DPB1 allelic sequences, locus-specific PCR primers were designed and applied to amplify the target sequence encompassing the entire exon 2 of HLA-DPA1 and HLA-DPB1. PCR products were purified with magnetic beads, and run through an ABI 3730 DNA sequencer. Genotypes were assigned with an Assign 3.5 SBT software.

RESULTSThe target sequences of HLA-DPA1 and HLA-DPB1 were both amplified with the PCR procedure. Little background and noise was observed in the derived sequences. Among 176 non-related healthy individuals, 4 HLA-DPA1 alleles with the frequencies of DPA1*02:02 (0.589) > DPA1*01:03 (0.284) > DPA1*02:01 (0.096) > DPA1*04:01 (0.031) were identified. In addition, 14 HLA-DPB1 alleles, including 4 common alleles (with a frequency of more than 5%, namely DPB1*05:01, DPB1*02:01, DPB1*04:01 and DPB1*02:02), 7 alleles with a frequency ranging from 1%-5% and 3 alleles with a frequency of less than 1% were identified. The results of HLA-DPB1 genotyping were all in accordance with the typing results derived from an Atria AlleleSEQR HLA-DPB1 kit.

CONCLUSIONA reliable technique has been established for simultaneous genotyping of HLA-DPA1 and HLA-DPB1, which may have a broad application in population and disease association studies.

Alleles ; Base Sequence ; DNA Fingerprinting ; methods ; Gene Frequency ; Genotype ; HLA-DP alpha-Chains ; genetics ; HLA-DP beta-Chains ; genetics ; High-Throughput Nucleotide Sequencing ; methods ; Humans ; Molecular Sequence Data ; Polymorphism, Genetic

Undifferentiated and differentiated 3T3-L1 adipocytes were treated with 100 ng/ml tumor necrosis factor-?(TNF-?),and peroxisome proliferator-activated receptor-?2 (PPAR-?2) mRNA expression and adiponectin secretion in cultured cells were measured.The results showed that TNF-?suppressed PPAR-?2 mRNA expression and adiponeetin secretion in 3T3-L1 adipocytes (P

Thirst study was purposed to explore the genetic polymorphism of Chinese Zhuang population at HLA-Cw locus by sequence based typing (SBT). A total of 150 unrelated blood samples from Chinese Zhuang population were subjected to sequencing at exon 2, 3 and 4 of HLA-Cw gene in both directions by using SBT technique established by our laboratory. The purified products of sequencing reaction were run by means of electrophoresis on the ABI 3730 DNA Sequencer and the assignment of HLA-Cw genotype was accomplished by using the Assign 3.5 software. The consensus sequence at exon 2, 3 and 4 of HLA-Cw gene for each sample was imported into the Assign 3.5 software. The results showed that 33.33% of tested samples could obtain an unique genotype, genotype in 63.33% of tested samples with ambiguous results could be assigned by ruling out the rare alleles according to the NMDP Rare Allele List File; however, the final genotype in rest 3.33% of the detected samples could be defined when subjected to further confirmatory testing by PCR-SSP. In this detection 16 HLA-Cw alleles were identified, the common alleles with a frequency of > 10% were Cw*0304 > Cw*0102 > Cw*0801 > Cw*0702. The value for gene diversity (GD) was 0.9297, The frequency for Cw*01, 03, 07, 08, 12, 14 (Cw 1 allele group) and Cw*02, 04, 05, 06, 15, 16, 17, 18 (Cw 2 allele group) was 0.8967 and 0.1032, respectively, which indicated that the Cw 1 allele group is the dominant ligand for KIR in Chinese Zhuang population. 51 genotypes were determined and the distribution of genotype frequency was in line with Hardy-Weinberg principle. It is concluded that the obtained HLA-Cw allele frequency and its distribution characteristics of Chinese Zhuang population can provide valuable data in the studies of anthropology and the association of HLA-Cw with disease.
Asian Continental Ancestry Group ; genetics ; Exons ; Gene Frequency ; Genotype ; HLA-C Antigens ; genetics ; Humans ; Molecular Sequence Data ; Polymorphism, Genetic ; Sequence Analysis, DNA

This study was aimed to develop and establish an efficient method for high through-put automatically extracting genomic DNA from EDTA-anticoagulated whole blood samples, and to utilize this method in routine rSSO HLA genotyping by luminex flow array assay, the genomic DNA was extracted automatically from 400 microl blood samples by using TECAN DNA workstation and 96-well plate with 2 ml volume per well. The yield and purity of each DNA sample was tested by UV-spectrophotometer, the integrity of these DNA samples were run electrophoresis on the agarose gel. Each DNA sample was subjected to PCR amplification and hybridization using One lambda rSSO HLA-A, -B and -DRB1 commercial kit, the fluorescent intensity for positive bead and negative bead hybridized with HLA-A, -B and -DRB1 PCR products were calculated and analyzed. The results showed that the mean yield and purity (A260/A280) of genomic DNA extracted from 400 microl whole blood samples were 3.217+/-0.715 microg and 1.710+/-0.103 respectively. The molecular weight was more than 15 kb in size and the fluorescent intensity for positive bead hybridized with HLA-A, -B and -DRB1 PCR products of each sample was >600 RFU, however, the fluorescent intensity for negative bead for each sample was <50 RFU. It is concluded that the highly qualified genomic DNA can be extracted automatically from blood samples of marrow-donors by using TECAN DNA workstation, and the extracted DNA samples are suitable for high through-put HLA genotyping by luminex flow array assay and other downstream transplant immunological and molecular biological experiments.
Biological Specimen Banks ; Bone Marrow ; DNA ; isolation & purification ; DNA Primers ; Genotype ; HLA Antigens ; genetics ; High-Throughput Screening Assays ; methods ; Humans ; Living Donors ; Nucleic Acid Hybridization ; methods ; Oligonucleotide Array Sequence Analysis ; methods

The objective of this study was to elucidate the correlation of killer immunoglobulin-like receptor (KIR) gene diversity with nasopharyngeal carcinoma (NPC) in the Chinese southern Han population. KIR genotyping of peripheral blood samples from 67 patients with NPC and 77 randomly-selected healthy controls was performed by PCR-SSP, the relative risk (RR) value was calculated by means of Wolf method. The results showed that the KIR2DL3 gene frequency in NPC patient group was significantly lower than that in healthy controls (χ²>3.84, p < 0.05, RR = 0.08), whereas the KIR2DS5 and KIR2DL5B gene frequencies in patient group were significantly higher than those in healthy controls (χ²>3.84, p < 0.05, RR > 1), the other KIR gene frequencies were no statistically different between two groups. It is concluded that the KIR2DL3, KIR2DS5 and KIR2DL5B genes may be correlated with pathogenesis of NPC in the Chinese southern Han population.
Adult ; Aged ; Asian Continental Ancestry Group ; genetics ; Case-Control Studies ; Female ; Gene Frequency ; Genotype ; Humans ; Male ; Middle Aged ; Nasopharyngeal Neoplasms ; genetics ; Receptors, KIR ; genetics ; Receptors, KIR2DL3 ; genetics ; Receptors, KIR2DL5 ; genetics

The aim of this study was to investigate the genetic polymorphism of Y-chromosome specific short tandem repeat (Y-STR) loci in Zhuang ethnic group of China. Nine Y-STR loci were amplified by single multiplex and the PCR products were detected by using ABI Prism(TM) 3100 DNA Sequencer. The allele frequencies and haplotype frequencies at 9 Y-STR loci were determined in a total of 85 unrelated male individuals from Zhuang ethnic group of China. The results indicated that in the 85 unrelated male individuals, except for the DYS426 locus with a low GD value, the GD values for other 8 Y-STR loci ranged from 0.4387 to 0.8129. A total of 70 haplotypes at 9 Y-STR loci were found, the haplotype diversity was 0.9926. It is concluded that the haplotype polymorphism of 9 Y-STR loci are highly polymorphic in Zhuang ethnic group and also significantly different from our previous reported data of unrelated male individnals in southern Chinese Han population.
Alleles ; China ; ethnology ; Chromosomes, Human, Y ; genetics ; Genetic Loci ; genetics ; Humans ; Male ; Microsatellite Repeats ; genetics ; Polymorphism, Genetic

A liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method was developed for the determination of budesonide in dog plasma. Budesonide and the internal standard triamcinolone acetonide were separated from plasma by alkalinized liquid-liquid extraction with ethyl acetate. Chromatographic separation was performed on a Capcell Pak C18 MG column with the mobile phase consisted of acetonitrile -5 mmol x L(-1) ammonium acetate (60:40, v/v) at a flow-rate of 0.50 mL x min(-1). A tandem mass spectrometer equipped with electrospray ionization source was used as detector and operated in the negative ion mode. Quantification was performed using multiple reaction monitoring (MRM) of the transitions m/z 489 --> m/z 357 and m/z 493 --> m/z 413 for budesonide and the internal standard, respectively. The linear calibration curves were obtained in the concentration range of 25.0-2000 pg x mL(-1). The lower limit of quantification was 25.0 pg x mL(-1). The intra- and inter-day relative standard deviation over the entire concentration range was less than 15%. The accuracy was in the range of -8.1% to -1.7% in terms of relative error. The method was applied to a pharmacokinetic study of budesonide controlled-release capsules in Beagle dogs. Maximal budesonide plasma level was observed after (3.5 +/- 3.3) h and the Cmax was (786 +/- 498) pg x mL(-1) after a single oral administration of 9 mg budesonide capsules, Cmax was increased to (2142 +/- 1515) pg x mL(-1) after multiple oral administration (9 mg x 5 d) of budesonide capsules. This method was selective and rapid, and the sensitivity was sufficient for the purpose of the pharmacokinetic study of budesonide controlled-release formulation.
Animals ; Anti-Inflammatory Agents ; blood ; pharmacokinetics ; Area Under Curve ; Budesonide ; blood ; pharmacokinetics ; Chromatography, Liquid ; methods ; Delayed-Action Preparations ; Dogs ; Male ; Sensitivity and Specificity ; Spectrometry, Mass, Electrospray Ionization ; Tandem Mass Spectrometry ; methods

OBJECTIVETo determine the origin of Rana temporaria for quality Oviduetus Ranae in the light of historical documents and modern researches on the classification of Rana temporaria chensinensis.

METHODWorks of Chinese meteria medica of all ages, related historical documents and reports from home and abroad on researches of R. temporaria chensinensis were consulted, sorted out, analyzed and summarized.

RESULTThe original Shange recorded in the works of Chinese meteria medica is R. temporaria chensinensis, which is the independent species, not one of species of European forest frogs. R. temporaria chensinensis is divided into 4 subspecies: R. temporaria chensinensis, Lanzhou, Kangding, and Changbaishan. The origin of R. temporaria is Changbaishan subspecies of R. temporaria chensinensis.

CONCLUSIONChangbaishan subspecies of R. temporaria chensinensis is determined as the origin for quality Oviduetus Ranae.

Animals ; Female ; History, 18th Century ; History, 19th Century ; History, 20th Century ; History, Ancient ; Materia Medica ; history ; isolation & purification ; Oviducts ; chemistry ; Rana temporaria ; anatomy & histology ; Ranidae ; anatomy & histology ; classification ; Species Specificity ; Terminology as Topic

This study was purposed to investigated and analyze the allelic frequency of a common allele HLA-C*08:22 in the southern Chinese Han population. A total of 32 samples with the C*08:01:01/08:22 ambiguous results previously identified in 163 unrelated southern Chinese Han population by routine sequencing based typing (SBT) at exons 2 - 4 of HLA-C gene were subjected to HLA-C SBT at exons 5 and 6 using our in-house method. Forty C*08:01:01-positive unrelated donor/recipient pair identified before the C*08:22 allele were officially nomenclatured and released by the World Health Organization (WHO) Nomenclature Committee for Factors of HLA System, were re-sequenced at exons 2 - 6 of HLA-C gene by our in-house SBT method. The allele assignment was accomplished with the Assign 3.5 SBT software. The results showed that three samples were identified as C*08:22-positive in the 32 samples with C*08:01:01/08:22 ambiguous results, the allele frequency of C*08:22 was 0.92% in the southern Chinese Han population. Retrospective analysis indicated that 2 donor/recipient pairs previously identified as C*08:01:01-positive were actually C*08:22-positive in the 40 tested donor/recipient pairs. It is concluded that the novel C*08:22 allele is the common allele in southern Chinese Han population, it can't be considered as rare allele and is ruled out for the samples with C*08:01:01/08:22 ambiguous results.
Alleles ; Asian Continental Ancestry Group ; genetics ; China ; Gene Frequency ; Genetics, Population ; HLA-C Antigens ; genetics ; Humans ; Sequence Analysis, DNA ; Unrelated Donors