BACKGROUND:During the research of ABO blood type antigen, the overwhelming majority samples of same ABO gene express a normal and same ABH antigen. But a certain amount samples with the same ABO genetic background show different antigen intensity expression as for different family or individuals. The ABO blood type has complex expression regulation mechanism. Analysis of ABO blood group serology and genetic background of these rare bi-specific AB phenotype specimens, and further studying on epigenetics may partly revealed ABO gene expression mechanism. OBJECTIVE:To study methylation of CpG island and explore the relationship between ABO gene promoter coding glycosyltransferase with dual donor specificity and ABH antigen expression. METHODS:Six samples detected as CisAB or B(A) phenotype were studied in this paper. The whole code sequences and promoter sequence of ABO gene were amplified respectively. The level of CpG methylation in promoter of ABO gene was further detected with bisulfite treatment method. RESULTS AND CONCLUSION:Among the six bi-specific AB phenotype samples, two previously-identified CisAB05/B(A)06 al eles with nt803C>G on the basis of B101 al ele sequence could be seen, and three additional methylated sites nt-33(30%), nt+27(50%) and nt+49(50%) were found between the two regions of CpG island in promoter of ABO gene. Two CisAB01 al eles with nt803C>G mutation on the basis of A101 sequence were found at nt-26C(10%). Other two B(A)04 al eles contained nt640A>G mutation on the basis of B101 sequence were found in the whole code sequences regions, and six additional methylated sites nt-33(10%), nt+16(50%), nt+57(60%), nt+59(60%), nt+68(60%) and nt+74(60%) were found between the two samples. No abnormity was identified in the promoter region of ABO gene. Our results indicated that the differential methylation levels in the CpG island of ABO gene promoter region may affect ABH antigens expression on the red cel membrane even if the samples had the same ABO genetic background.

Objective To analyze the relationship between traditional & emerging risk factors and the pathogenesis of atherosclerotic renal artery stenosis (ARAS) in different gender patients.Methods Selective renal artery angiography was performed immediately after routine coronary angiography in 2060 patients with suspected coronary artery disease.Traditional & emerging risk factors were recorded and compared in different gender patients with ARAS.Results Among the patients with ARAS, smoking (59.18％ vs.12.50％, x2 =17.47,P＜0.01) and diabetes(28.57％ vs 46.87％, x2 =5.64, P＜0.05) compared with both genders were significantly differences.There were significant differences in high density lipoprotein cholesterol, ApoA1, ApoB/ApoA 1, HbA1C and Hcy (t =2.62,2.07,-2.83,-2.37,3.74;P＜ 0.05) in the ARAS patients compared with both genders.Conclusion The main risk factors in different gender patients with ARAS were different.It is important to adopt appropriate control strategies.

Objective To establish a sandwich ELISA for early detection of HIV antigens using a mixture of monoclonal antibodies (McAb). Methods The ascites McAbs (anti-HIV-1 p24, anti-HIV-1 gp41, anti-HIV-1 gp120 and anti-HIV-2 gp36) were purified by the SAS and the affinity chromatography,and then were labeled with HRP by sodium metaperiodate. The establishing of sandwich ELISA for detecting the single HIV antigen and the tests of specificity and sensitivity of these systems were performed in advance.A proper ratio mixture of four screened McAbs was used as the capture antibody and a proper ratio mixture of four labeled antibodies was used as the detecting antibody. The method of using sandwich ELISA to detect HIV antigens was set up with these McAbs. Results The sensitivity of this method detecting HIV antigens are:0.625 pg/ml HIV-1 p24, 6.25 ng/ml HIV-I gp41,6.25 ng/ml HIV-I gp120 and 9.25 ng/mi HIV-2 gp36 in mixed HIV antigens. Conclusion The method of using several McAbs mixture in sandwich ELISA detecting HIV antigens was established an excellent sensitivity, which provides a novel idea for early detec-ting the HIV antigen.

Objective To study the molecular genetic background of Bel subtype at ABO blood group.Methods Three samples and fifteen samples were diagnosed as Bel subgroup and normal control samples by serological test,respectively.The extracted DNA was genotyped by sequence specific primer- polymerase chain reaction foilowed by sequencing for Exon6 and exon7 at ABO locus and clones were sequenced.Results A novel Bel variant allele(GenBank EF117687) was identified in a Bel individual.The Bel allele was different from the regular B101 allele by single 952G>A missense mutation in exon7.resulting in an amino acid subsfitution of Val for Met at 318 locus.No mutations were detected in the fifteen control samples and the other two Bel allele samples.Conclusions The mutation position was fimt found to lie on coding region of ABO gene behind nucleotide 930.The mutation of G952A in the al,3 galactosyhransferase gene may be one of the molecular genetic basis of Bel ohenotype.

Objective To investigate serological blood typing of the ABO locus which contradict to general law of inheritance in parentage,and the underlying reasons for severe hemolytic disease of newborn(HDN).Methods To research the family whose newborn is AB phenotypes,mother is O phenotypes and father is AB phenotypes.The familiy were genotyped by parentage tests, serological tests,PCR-SSP and direct DNA sequencing at exons 6 and 7 of ABO gene.At the salne time,HDN was detected by micro column gel Coombs (MGCT), and the primary fingerposts of the routine blood tests. Biochemical tests were dynamically observed.Results The results of parentage tests showed that three-generation pedigree have parent-child relationship. The red blood cell(RBC)of this AB phenotypes of this family members strongly agglutinated(4+)with diverse monoelonal anti-A and anti-B antibodies,and their serum did not contain anti-A and anti-B antibodies in blood anti-typing.PCR-SSP can not detect their A and B gene,but DNA sequencing at exons6 and 7 of ABO gene revealed that it had the B(A)803C→G mutation.Conclusions The genetm basis of this parentage are B(A)803G blood gene which harbored both A and B difunctionality of glyeosyhransferases.This was the first report that severe HDN resulting from a large number of A and B antigens in RBC of B(A)phenotype of a newborn,which has clinical significance on ABO locus.

Objective To identify novel ABO allele in Chinese population. Methods The ABO blood group was tested by serological method, and then genotyped by sequence-specific primer (PCR-SSP) , gene cloning and sequence analysis. Results A healthy blood dornor who was diagnosed as having A2 subgroup and A2O1genotype was subjected to ABO gene cloning and sequence analysis. The haplotype-specific sequence analysis indicate that two single-base deletions, where G-deletion at nucleotide position 261 and A-deletion at nucleotide position 496 were determined in the O1 allele. The nucleotide sequence of the novelO1 allele were identical to ABO 0101 allele except for A-deletion at nucleotide position 496 in exon7 of ABO locus. Conclusion We defined this 0 allele as a novel O1 variant allele, and its registered number by GenBank is AY374123.

Background Voriconazole is the traditionally used antifungal agent,but its ophthalmic form is unsatisfactory.A novel ophthalmic drug delivery system with biomedical devices may be of promising for the prognosis of fungal keratitis.Objective This study was to investigate the sustained release,therapeutic effect and biocompatibility of effect and quaternized chitosan functionalized with carboxylated graphene and nano-silver and voriconazole (CS-ETA/Ag/GO/Vor) for fungal keratitis.Methods This study complied with the Regulations for the Administration of Affair Concerning Experimental Animals of State Science and Technology Commission.Two hundred and ten SPF female C57BL/6 mice were selected with the age 6-8 weeks for the biocompatibility experiment (30 mice) and therapeutic observation of CS-ETA/Ag/GO/Vor (180 mice).CS-ETA/Ag/GO and CS-ETA/Ag/GO/Vor were attached on the normal corneas of mice and compared with the normal mice to assess the histopathological changes.Aspergillus fumigatus-infected mouse models were established in the left eyes of 180 mice by intrastromally injection of 2.0 μl Aspergillus fumigatus suspension with the density of 5 × 107 CFU/ml,then the mice were randomized into the model control group,CS-ETA/Ag/GO group and CS-ETA/Ag/GO/Vor group,and the corresponding membrane were attached the central corneas in different groups.In 1 day,3,5,7 days after modeling,the corneas were examined under the slit lamp microscope and scored,and corneal sections were prepared for the histopathological examination.Fungal activity was confirmed by plate counts,and real-time PCR was employed to assay the relative expressions of interleukin-1β (IL-1β) mRNA and tumor necrosis factor-α (TNF-α) mRNA in the corneas.Results No morphological abnormality was seen in the corneas in the normal control group,CS-ETA/Ag/GO group and CSETA/Ag/GO/Vor group.Corneal inflammatory score was significantly lower in the CS-ETA/Ag/GO/Vor group in various time points,with a significant differences among the groups and time points (Fgroup =237.29,P=0.00;Ftime =260.33,P=0.00).The edema,necrosis or perforation of cornea were seen in the model control group,and slighter inflammatory response in the CS-ETA/Ag/GO group,and corneal edema was gradually disappear in the CS-ETA/Ag/GO/Vor group.The corneal fungal loads were highest in the model control group and lowest in the CS-ETA/Ag/GO/V or group,with significant differences among the three groups and various time points (Fgroup =113.15,P =0.00;Ftime =126.52,P=0.00).The relative expressions of IL-1β mRNA and TNF-α mRNA in the corneas peaked in the fifth day after modeling in all of the three groups,and the expression levels of IL-1β mRNA and TNF-α mRNA in the corneas were lowest in the CS-ETA/Ag/GO/Vor group,showing significant differences among the groups and time points (IL-1β:Fgroup =189.90,P =0.00;Ftime =108.56,P =0.00;TNF-α:Fgroup =82.55,P =0.00;Ftime =44.36,P =0.00).Conclusions CS-ETA/Ag/GO/Vor delivery system plays an anti-fungal activity in fungal keratitis by the synergistic effect of voriconazole and Ag+.In addition,CS-ETA/Ag/GO/Vor appears to have a good safety after topical application.

Objective:To study the regulatory effects of Ligustrazine Hydrochloride(LHC)on tumor-induced immunosuppression by Colon26 cells in vitro.Methods:Colon26 cells were cultured for 48 h in the presense of LHC and either the cell fraction or the cultural supernatants was collected,with the untreated Colon26 cells as control for the study.The down-regulating effects of LHC on tumor immunosuppressions (including the suppressed NK killing and ConA induced transformation of murine spleen cells detected by MTT,and the reduced expression levels of IL-2R?,CD3?+?+ and CD3?-?+ detected by FCM) were determined.The concentrations of immunosuppressive cytokines,including TGF-?1,VEGF,IL-4,IL-6 and IL-10,in the supernatants were analyzed by quantitative ELISA.The relationship among the down-regulatory effects of LHC on secretion immunosuppressive cytokines and tumor immunosuppressions were evaluated by multiple linear regression analysis.Results:All of the cytokines assayed were found in the supernatant of Colon26 treated without LHC,in which TGF-?1 was the highest,and the significant inhibition of five immune functions mentioned above was showed.To the Colon26 treated by LHC,the concentrations of TGF-?1,IL-6 and IL-10 in the first re-cultured supernatant and its inhibition of five immunol functions decreased greatly.The concentrations of TGF-?1 and IL-6 in the second re-cultured supernatant and its inhibitions of transformation,CD3+?+ and CD3-?+resumed highly.The positive correlations existed between TGF-?1 and inhibition of immunol functions except for transformation,between IL-6 and inhibition of transformation or CD3-?+,between IL-10 and inhibition of NK killing or IL-2R? or CD3+?+,respectively.Conclusion:LHC can exert down-regulate effects on Colon26 secretion of immunosuppressors and its tumor immunosuppression.Reducing tumor immunosuppression of Colon26 through decreasing its secretion of immunosuppressors should be one of anti-tumor mechanisms of LHC.

OBJECTIVETo detect the mutation of PORCN gene in a patient with focal dermal hypoplasia and study the genotype-phenotype correlation.

METHODSPeripheral blood samples were obtained from the family members and control subjects. PCR was carried out to amplify all the exons and adjacent splice sites of PORCN gene and mutation was detected by bidirectional sequencing.

RESULTSA G149C mutation was found at exon 2 of the PORCN gene in the patient, which caused a change from Alanine to Proline at codon 38 (A38P). The patient presented mild clinical manifestations.

CONCLUSIONA new missense mutation (A38P) in the PORCN was detected in the patient, which maybe one of the molecular mechanisms in the pathogenesis of the disease. The relationship between G149C genotype and moderate phenotype might be attributed to the influence of A38P missense mutation towards the corresponding protein, which is different from previous results.

Acyltransferases ; Base Sequence ; Child ; DNA Mutational Analysis ; Female ; Focal Dermal Hypoplasia ; genetics ; pathology ; physiopathology ; Humans ; Membrane Proteins ; genetics ; Mutation ; genetics

OBJECTIVETo study the gene polymorphism of the Auberger antigens in Lutheran blood group system in Chinese population and establish a stable, accurate molecular method detecting Auberger antigens.

METHODSPeripheral blood samples from 162 randomly collected and unrelated volunteer blood donors were directly sequenced for the exon 12 at the gene locus of Auberger antigens. PCR products with novel nucleotide were further investigated by restriction fragment length polymorphism (RFLP) analysis.

RESULTSAuberger genotypes in the 162 Chinese individuals were obtained: Au(a+ b- )(nt1615A) was found in 119 individuals, Au(a+ b+ ) (nt1615A/G) in 40 individuals and Au(a- b+ ) (nt1615G) in 3 individuals. The allele frequencies of the Au(a) and Au(b) were 0.8580 and 0.1420, respectively. An individual with homozygous Au(a) genotype had a nucleotide mutation (1595 G to T). The mutation was confirmed by digesting the DNA with Hha I.

CONCLUSIONThe distribution of gene polymorphism of Auberger antigens in a Chinese population was investigated and obtained. And a molecular method determining the Auberger antigen was established. A novel Lutheran allele was deposited in GenBank (accession number EU260043).

Amino Acid Sequence ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; DNA Mutational Analysis ; Female ; Gene Frequency ; Haplotypes ; Humans ; Lutheran Blood-Group System ; chemistry ; genetics ; Male ; Molecular Sequence Data ; Polymorphism, Genetic