Sample preparation and separation is a very crucial and complicated process in proteomics research.Based on the actual condition of university and students,this article explored the way to improve the teaching quality of sample preparation and separation in proteomics course for medical post-graduates aiming at cultivating and enhancing the students' scientific thought,making them have a better understanding of the rules,technologies,strategies in sample preparation and separation procedure.

OBJECTIVETo investigate the antithrombin (AT) activity (AT: A) and AT antigen (AT: Ag) level in a Chinese family with type I antithrombin (AT) deficiency, and to explore the molecular mechanism of AT deficiency.

METHODSImmuno-nephelometry and chromogenic assay were used to detect the plasma level of AT: A and AT: Ag, respectively. Genomic DNA was isolated from the peripheral blood, and all the seven exons and exon-intron boundaries of AT gene were amplified by PCR and direct sequencing.

RESULTSThe plasma levels of AT: A and AT: Ag of the proband were 45% and 97 mg/L, respectively, which led to a type I AT deficiency. A heterozygous T to A mutation was found at nucleotide 9833 in exon 5 resulting in a Tyr363Stop nonsense mutation. The sequencing results from the pedigree indicated that four other members also had this mutation.

CONCLUSIONThis heterozygous nonsense mutation of T9833A in exon 5 resulting in venous thrombosis is a novel genetic defect of hereditary AT deficiency, which has not been described before.

Antithrombin III Deficiency ; genetics ; Antithrombins ; genetics ; Blood Coagulation Tests ; Female ; Humans ; Male ; Mutation ; Pedigree ; Polymerase Chain Reaction ; Sequence Analysis, DNA

OBJECTIVETo investigate the antithrombotic mechanisms of holothurian glycosaminoglycan (GAG) extracted from sea cucumber.

METHODSHuman endothelial cell line EA. hy926 cells were treated with 10 mg/L GAG or 10U/mL unfractionated heparin (UFH) by short-term (15 min - 2 h) and longer-time incubation (6 h - 48 h). Different doses of GAG were used to stimulate EA. hy926. Released free tissue factor pathway inhibitor(TFPI) was determined by ELISA assay. TFPI expression was investigated by immunofluorescent method and TFPI mRNA level by real-time PCR. In a 96-wells microtitre plate, pooled normal plasma containing different concentrations of GAG was allowed to clot by addition of thrombin and calcium chloride, fibrinolysis was induced by addition of t-PA. TRR (TAFI-related retardation of clot lysis) was used to assess thrombin-activatable fibrinolysis inhibitor(TAFI) functional activity.

RESULTSGAG increased TFPI synthesis, expression and secretion in a dose- and time dependent manner. GAG at low concentrations could lengthen while at intermediate concentrations could shorten clot lysis times significantly as compared to control values. TRR was dose-dependently decreased on addition of GAG.

CONCLUSIONSGAG increases TFPI synthesis, expression and secretion of endothelial cells. GAG at intermediate concentrations significantly affects clot stability of a developing clot by means of diminishing TAFI activation.

Animals ; Carboxypeptidase B2 ; antagonists & inhibitors ; Cell Line ; Dose-Response Relationship, Drug ; Endothelial Cells ; drug effects ; metabolism ; Glycosaminoglycans ; pharmacology ; Heparin ; pharmacology ; Holothuria ; Humans ; Lipoproteins ; biosynthesis ; genetics ; RNA, Messenger ; genetics ; Tissue Extracts ; pharmacology

OBJECTIVETo investigate the molecular mechanism of haemophilia B caused by the novel mutation of Arg327Ile (R327I) in FIX gene.

METHODSThe R327I, R327Ala(A), R327Lys(K), R327Asn(N) and a replacement mutant (FIXβFVII), in which FIX β strand 324-329 was replaced by that of FVII 298-303, expression plasmids were constructed with site-directed mutagenesis method based on the wild-type (WT) FIX expression plasmid. The HEK293 cell was transiently transfected, then the activity of FIX (FIX:C) was assayed by one stage method in the conditioned medium, while the FIX:Ag in both the conditioned media and the cell lysates was measured by ELISA. The molecular weight and the semi-quantity of expressed FIX were analyzed by Western blot. Fluorescent protein expression plasmid was constructed to investigate the synthesis and secretion of the FIX R327I mutation in the viable cells.

RESULTSFIX:C of the R327I mutant protein was 4.49% of the level of the WT in the conditioned medium, and the FIX:Ag of the R327I mutant protein in the conditioned medium and the cell lysates was 31.02% and 129.29% compared to that of WT, respectively. The mutation was characterized as cross-reaction material reduced (CRMR). The viable cell fluorescent assays showed that the R327I protein was more in both the viable cells and in lysosome than that of WT. The FIX:C of the R327A, R327K, R327N and FIXβFVII mutants was reduced compared to that of WT, the reduction of FIX:C of FIXβFVII was the most significantly amount among all the mutants in medium. FIX:Ag of all the mutants in the medium, except that the R327K increased, was reduced. The result of Western blot showed that the molecular weight of R327I protein was the same as that of WT, but the amount of the protein was much less compared with WT in the conditioned medium.

CONCLUSIONThe abnormal synthesis and secretion as well as the abnormal function of the R327I mutant protein causes haemophilia B. The residue of R327 as well as the β strand domain of R327 located play important roles of the specific function of FIX.

Factor IX ; genetics ; HEK293 Cells ; Hemophilia B ; genetics ; pathology ; Humans ; Mutagenesis, Site-Directed ; Mutation ; Transfection

OBJECTIVETo identify the antithrombin (AT) phenotype and gene mutation of a kindred with hereditary antithrombin deficiency.

METHODSPlasma AT activity and AT antigen level of the propositus and his kindred members were determined with chromogenic substrate method and immunoassay, respectively. All the seven exons and intron-exon boundaries of antithrombin gene were analyzed by PCR and direct sequencing of amplified PCR products from the propositus.

RESULTSThe propositus AT antigen level was normal but his AT activity was only 65% of normal value suggesting that he had type II AT deficiency. A heterozygous G13830A mutation in exon 6 resulting in Arg393His missense mutation in his AT polypeptide was identified in the propositus. The same phenotype and gene mutation were found in other 3 kindred members.

CONCLUSIONThe type II AT deficiency found in this kindred is caused by heterozygous G13830A mutation in AT gene.

Adult ; Antithrombin III ; genetics ; metabolism ; Antithrombin III Deficiency ; genetics ; Heterozygote ; Humans ; Male ; Mutation ; Pedigree

2 in some patients with the loss of high and medium sized vWF multimers in plasma.Eight patients with vWD were identified, wherein two were characterized as type 1,4 as type 2A and 2 as type 3 respectively.Conclusion The panel of tests is suitable for diagnosis and classification of vWD.

Objective To estimate correlation between phonetically balanced maximum(PB max)and pure tone auditory threshold in auditory neuropathy(AN)patients.nethods 0ne hundred and six ANpatients were identified using multipie criteria including PB max,a metric for speech recognition,pure tone auditory threshold.acoustic emission test.distortion products otoacoustic emission(DPOAE) and auditory brainstem response(ABR).SPSS statistical software was used to estimate the Pearson's correlation between PB max and pure tone auditory threshold and to test whether pure tone auditory threshold,or auditory configuration had a significant impact on PB max.Results Even the patients had the same or similar values for pure tone auditory threshold or auditory configuration.varied values of PB max were found in two hundreds and twelve ears for 106 patients.Analysis of the data for 106 patients revealed a negative correlation(r=-0.602,P<0.01) between PB max and pure tone auditory threshold,i.e.hearing loss at a mild relates to a lower PB max.By using analysis of variance(ANOVA)method,it Was found that both pure tone auditory threshold and auditory configuration had a significant(P<0.01)impact on the patients' PB max.Conclusions This analysis implicated the promise and potential of pure tone auditory threshold and auditory configuration for predicting PB max of the AN patients,and improving the diagnosis of AN.

OBJECTIVETo analyze intron 1 and 22 inversions in factor VIII (FVIII) gene in hemophilia A (HA) patients and and their families and to investigate the correlation between intron inversion and FVIII antibody.

METHODSAll patients were detected FVIII: C and FVIII antibody. In addition, 81 unrelated HA patients were directly detected by multiplex PCR and long-distance PCR for intron 1 and 22 inversions in FVIII gene. Pedigree investigation for some patients were conducted.

RESULTSIn 81 unrelated HA patients, 3 severe cases were found intron 1 inversion which accounted for 4.6% of total 65 severe cases. Of the 3 cases, one was FVIII antibody positive. Two female family members of a intron 1 inversion patient were identified as one carrier and one non-carrier. Twenty five of 65 (38.5%) severe cases were found intron 22 inversion. Of the 25 cases 1 was FVIII antibody positive. Nine female members in 5 HA families which had patients with intron 22 inversion were identified as 7 carries and 2 non-carriers.

CONCLUSIONBesides intron 22 inversion, intron 1 inversion was another important molecular defect in resulting in severe HA. Intron inversion analysis can also be used for deviation rectification of experiment grouping in HA patients. Intron 1 and 22 inversions may be one of the higher risk factors for resulting in FVIII antibodies.

Chromosome Inversion ; Chromosomes, Human, X ; Factor VIII ; genetics ; Female ; Hemophilia A ; genetics ; Humans ; Introns ; Male

OBJECTIVETo explore F (13) A gene mutation in a pedigree with hereditary coagulation factor XIII (FXIII) deficiency.

METHODSThe FXIII deficiency was diagnosed by clot solubility test and other standard laboratory clotting tests. All exons, exon-intron boundary sequences of F(13) A gene were amplified by PCR and the products were sequenced directly. Any mutation identified by direct sequencing was confirmed by reverse sequencing. The mutation identified in the proband was screened in the family members.

RESULTSThe assays of PT, Qiulan, fibrinogen leveling, platelet counts, bleeding time were normal and the clot solubility test was positive in the proband. The homozygous deletion of 33 nucleotides (127067de133) in exon 10 of F(13) A gene which resulted in deletion of 11 amino acids in FXIIII A protein with 720aa residues was identified in the proband. Family studies showed that the mutation was inherited from the parents both of whom carried the heterozygous deletion mutation.

CONCLUSIONThe homozygous 127067de133 mutation of F(13) A gene is responsible for the disorder of the pedigree.

Adolescent ; Factor XIII ; genetics ; Factor XIII Deficiency ; genetics ; Heterozygote ; Homozygote ; Humans ; Male ; Pedigree ; Sequence Deletion

OBJECTIVETo identify the relationship between coagulation factor V (FV) gene single nucleotide polymorphisms (SNPs) and venous thromboembolism (VTE).

METHODSThe FV clotting activity (FV: C) and FV antigen (FV: Ag) in plasma of VTE group (111 patients) and normal control (110 patients) were detected using one-stage clotting assay and ELISA, respectively. Five pairs of primers of the F V polymorphisms including Asp79His, Arg306The, Arg306Gly, Arg506Gln and Ile359The/His1299 Arg were synthesized and amplified by PCR. The PCR products were digested by restriction enzyme using PCR-RFLP. The detected polymorphisms were confirmed by direct sequencing. The samples containing the polymorphisms were screened for coding regions of all F V exons with direct sequencing.

RESULTSThe plasma levels of F V: C and F V: Ag of VTE group and normal control were (106.9 +/- 28.0)%, (110.4 +/- 33.3)% and (102.4 +/- 30.9)%, (102.1 +/- 24.1)%, respectively. The plasma level of FV: Ag was significantly different between VTE group and normal control. However, there was no difference in F V: C levels. Polymorphisms for the fore mentioned 5 primer pairs were not found in either patients or normal controls. Polymorphism of His1299Arg was identified in 5 patients with VTE and 3 normal controls. And these 5 cases also combined Met1736Val polymorphism, 3 of them combined another Asp2194Gly polymorphism.

CONCLUSIONThe higher plasma level of F V: Ag contribute to venous thromboembolism. There is no relationship between polymorphisms of Asp79His, Arg306The, Arg306Gly, Arg506Gln, Ile359The and venous thromboembolism in Chinese studied. Polymorphism His1299Arg is higher in VTE group than in normal control, but has no statistical difference. Polymorphisms of His1299Arg, Met1736Val and Asp2194Gly are linked disequilibrium in Chinese Han population.

Factor V ; genetics ; Female ; Gene Frequency ; Humans ; Linkage Disequilibrium ; Male ; Middle Aged ; Polymorphism, Single Nucleotide ; Venous Thromboembolism ; genetics