Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) followed by mass spectrometry (MS) is the most widely used method of protein resolution and identification. But it exist shortcomings. In this paper introduce some novel methods for protein resolution and identification,including the methods by high-liquid chromotography,capillary isoelectric focusing,capillary electrophoresis or 1D and 2D microcapillary chromotagraphy.

Objective To approach the effect of down regulation of fast glucose damge standard on endothelial funtion. Methods The FBS 、serum lipid、 uric acid of 54101 normal subjects were detected, those subjects were divided by FBG value into four groups: A B C D ,the value of TC、 TG、 uric acid in group B were higher than which in group A, lower than in group C. The value of PBG、FINS、PINS、ET、NO of 655 subjects choiced randomly were detected .At the same time the endodermis dependence angiectasia funtion mediated by bloodstream and the no-endodermis dependence angiectasia funtion mediated by glycerol trinitrate were detected . Results The value of group B was lower than which in group A, having no statistical meanings compared with group C、D;the no-endodermis dependence angiectasia funtion mediated by glycerol trinitrate of group B was higher than that of group C, that of group C was lower than group A, the comparison of other two groups has no statistical meanings. Conclusions Down regulation of fast glucose damge standard can effect on the endothelial funtion.

Objective:To explore the influence factors on amplification of single cell duplex-nested PCR.Methods:The mutational loci region CD41-42 and IVS-Ⅱ654 of ?-globin gene were amplified by duplex-nested PCR with different combination of primers concentration, different Taq DNA polymerases, different neutralization buffers and with or without predenaturation at 98 ℃ before the PCR amplification in single lymphocyte or single blastomere, thus, to investigate the influence of these factors on the amplification efficiency of PCR.Results:TaKaRa EX Taq was the most efficient Taq DNA ploymerase among different Taq DNA ploymerases; primer pair R1+F1 at final concertration of 0.25 ?mol/L and R2+F2 at 0.3 ?mol/L were the most efficient ones in amplification among different combinations of primers concentrations; the amplification efficiency in neutralization buffer-1 (200 mmol/L Tricine) was obviously higher than that of neutralization buffer-2 (900 mmol/L Tris-HCl, pH 8.3/300 mmol/L KCl/200 mmol/L HCl)(P0.05). Conclusion:There were remarkable differences of the amplification efficiency of single cell duplex-nested PCR while using different combination of primers concentrations, different Taq DNA polymerases, different neutralization buffers. However, predenaturation at 98 ℃ before the single cell PCR amplification could not improve the PCR amplification efficiency

Objective To evaluate the fallopian tube patency with transvaginal real-time three-dimensional hysterosalpingo-contrast sonography (3D-HyCoSy) in diagnosis of infertility.Methods Taking laparoscopy and dye test as the reference standard,a total of 126 infertile women underwent both transvaginal real-time 3D-HyCoSy with SonoVue and laparoscopy.Bilateral fallopian tubes were dynamic observed.Results Transvaginal real-time 3D-HyCoSy showed that in clinical suspected infertility patients,125 fallopian tubes were patent,81 fallopian tubes were narrow and circuitous,and 37 fallopian tubes were obstructed.Among all the patients,60 cases (115 fallopian tubes) underwent laparoscopy with chromopertubation.The results showed that 10 fallopian tubes were patent,73 fallopian tubes were narrow and circuitous,and 32 fallopian tubes were obstructed.Taking laparoscopy and dye test as the reference standard,107 fallopian tubes of the 60 cases were diagnosed correctly,and the coincidence rate of 3D-HyCoSy and laparoscopy and dye test was 93.0％ (107/115).Taking laparoscopy and dye test as the gold standard,the accuracyof transvaginal real-time 3D-HyCoSy in diagnosis of fallopian tubes patency or obstruction was 93.0％(107/115),the sensitivity was 94.3％ (99/105),the specificity was 80.0％ (8/10).Conclusions Tansvaginalreal-time 3D-HyCoSy can provide dynamic,real-time and three-dimensional display of the fallopian tubes.Itcan get more accurate diagnostic information,so it's an important method.3D-HyCoSy is a safe and non-invasivemethod,so it plays an important clinical role in the screening,diagnosis and treatment of gynecological diseases.

ObjectiveTo observe the affection of cilostazol combined with valsartan in treating patients with early diabetic nephropathy on serum homocysteine and cystatin C,MethodsNinety-six cases with early diabetic nephropathy were divided into two groups by random digits table method with 48 cases each:observation group was treated with cilostazol and valsartan,control group was treated with valsartan alone.The effect and the levels of 24 h urinary albumin,serum homocysteine and cystatin C before and after treatment in two groups were compared.ResultsThe total effective rate in observation group [85.4％(41/48) ] was significantly higher than that in control group [66.7％(32/48) ](P ＜ 0.05 ).The levels of 24 h urinary albumin,serum homocysteine and cystatin C were significantly decreased after treatment of three weeks in observation group and control group [ (125.48±13.76) mg,(9.25±3.52)p mol/L,(7.82±2.14) mg/L and (168.38±15.43) mg,(13.72±4.23) μ mol/L,(9.57±2.85) mg/L vs.(279.31±21.52) mg,(18.52±6.14) μ mol/L,( 13.25±3.79) mg/L and (275.24±19.31 ) mg,( 18.48±6.12) μ mol/L,( 13.19±3.76)mg/L](P＜ 0.05).And the levels of 24 h urinary albumin,serum homocysteine and cystatin C after treatment of three weeks in observation group were obviously lower than those in control group(P ＜ 0.05).Conclusions Cilostazol combined with valsartan in treating patients with early diabetic nephropathy can improve clinical effect obviously,degrade the level of urinary albumin,homocysteine and cystatin C.Therefore,it deserves to be applied in clinic.

OBJECTIVETo discuss the genetic diagnosis of congenital adrenal hyperplasia (CAH) and investigate the resource of gene mutations in CAH.

METHODEnzymatic methods with restriction endonucleases that specifically recognized the mutation sites were used to detect the gene mutations in patients with CAH and their relatives. Polymerase chain reaction and direct sequencing were used to identify the mutations in 21-hydroxylase gene, and short tandem repeat (STR) typing was used to determine the sources of the mutations.

RESULTSOne CAH patient had two known mutations in 21-hydroxylase gene, namely the I2g and I172N mutations. The former mutation was inherited from the biological mother and the latter was not inherited.

CONCLUSIONThe 9 common mutations of CAH are also the hotspots for new mutations.

Adrenal Hyperplasia, Congenital ; diagnosis ; genetics ; Gene Deletion ; Genotype ; Humans ; Mutation ; Point Mutation ; Polymerase Chain Reaction ; Steroid 21-Hydroxylase ; genetics

OBJECTIVETo study the clinical application of denature high performance liquid chromatography (DHPLC) technique on mutation screening and prenatal diagnosis for Wilson's disease (WD).

METHODSGenomic DNA of the probands with Wilson's disease and their parents from 6 families was subjected to polymerase chain reaction (PCR) for the 21 exons and the 5' untranslated region of ATP7B gene. Mutation screening of the PCR products was performed by DHPLC. The abnormal peaks were confirmed by further sequencing analysis. Based on the successful gene diagnosis for the patients, prenatal diagnosis was performed in 4 families, including 1 twin and 3 singletons.

RESULTSFive disease-causing mutations and 8 polymorphisms were found in the 6 probands by DHPLC and sequencing. The parents were carriers with the same mutation as their affected children. Prenatal diagnosis showed that two pregnancies were abnormal, including a twin pregnancy with compound heterozygote for Arg778Leu and IVS4-1G>C mutation, and a single pregnancy with a compound heterozygote for Ser975Tyr and Pro992Leu mutations. These two pregnancies were terminated after genetic counseling. Another two pregnancies included a singleton carrier with Ser975Tyr mutation and a normal genotype fetus, respectively. These two pregnancies were continued and the babies were healthy.

CONCLUSIONDHPLC is a powerful tool in prenatal diagnosis as well as in postnatal diagnosis.

Adenosine Triphosphatases ; genetics ; Cation Transport Proteins ; genetics ; Chromatography, High Pressure Liquid ; methods ; Copper-transporting ATPases ; DNA Mutational Analysis ; Exons ; genetics ; Female ; Fetal Diseases ; diagnosis ; genetics ; Genetic Testing ; methods ; Hepatolenticular Degeneration ; diagnosis ; genetics ; Humans ; Male ; Mutation ; Pedigree ; Pregnancy ; Prenatal Diagnosis ; methods

OBJECTIVETo investigate and establish the gene diagnosis methods for the frequent mutations of iduronate-2-sulphatase(IDS) gene in mucopolysaccharidosis type II patients.

METHODSpolymerase chain- reaction-single strand conformation polymorphism PCR-SSCP) analysis was applied to detect the mutations of exons 3, 8 and 9 which were hot spots in the iduronate-2-sulfatase gene; DNA sequencing was applied to analyze the mutations which had been detected by PCR-SSCP; PCR-restriction fragment length polymorphism (PCR-RFLP) was applied to detect the results of DNA sequencing.

RESULTSObvious and abnormal bands in exon 9 of the IDS gene were found by applying PCR-SSCP; the mutation(C1672T) of exon 9 was found in the patient through DNA sequencing, which led to amino acid replacement(R468W); the PCR-restriction enzyme digestion showed that only one band(554 bp) appeared in the patient, but there were two bands (257 bp and 297 bp) in his parents, and it verified the results of sequencing analysis.

CONCLUSIONPCR-SSCP analysis, DNA sequencing analysis and PCR-restriction enzyme digestion are effective methods for MPS II diagnosis. Combined applications of these methods can verify and complement each other and improve the accuracy of diagnosis.

Amino Acid Substitution ; Base Sequence ; Child ; China ; Codon, Nonsense ; DNA ; chemistry ; genetics ; DNA Mutational Analysis ; Humans ; Iduronate Sulfatase ; genetics ; Male ; Mucopolysaccharidosis II ; enzymology ; genetics ; Mutation ; Point Mutation ; Polymorphism, Single-Stranded Conformational

Chromosomal translocation is a kind of common chromosomal abnormality. The carriers with chromosomal translocation could have more chance of normal pregnancy with the help of fluorescence in situ hybridization(FISH). This is a review aimed at analyzing the meiosis types of the translocation chromosome. The strategy of preimplantation genetic diagnosis for the carriers with chromosomal translocation is also discussed.
Humans ; In Situ Hybridization, Fluorescence ; methods ; Meiosis ; genetics ; Preimplantation Diagnosis ; methods ; Translocation, Genetic ; genetics

OBJECTIVESTo evaluate the relationship between microdeletion or mutation on the Y chromosome and Chinese patients with idiopathic azoospermia and severe oligozoospermia and to establish a molecular detection method.

METHODSMicrodeletion or mutation detection at the AZFa (sY84 and USP9Y), AZFb, AZFc/DAZ and SRY regions of the Y chromosome. Seventy-three azoospermia and 28 severe oligozoospermia patients were evaluated using PCR and PCR-SSCP techniques.

RESULTSTwelve of 101 patients (12%) with the AZFc/DAZ microdeletion were found, including 8 with azoospermia (11%) and 4 with severe oligozoospermia (14.3%), and 1 patient had a AZFb and AZFc/DAZ double deletion. No deletions in the AZFa or SRY regions were found. No deletions in AZFa, AZFb, AZFc/DAZ or SRY regions were found in 60 normal men who had produced one or more children.

CONCLUSIONSMicrodeletion on the Y chromosome, especially at its AZFc/DAZ regions, may be a major cause of azoospermia and severe oligozoospermia leading to male infertility in China. It is recommended that patients have genetic counseling and microdeletion detection on the Y chromosome before intracytoplasmic sperm injection.

Chromosome Deletion ; Humans ; Male ; Oligospermia ; genetics ; Sperm Injections, Intracytoplasmic ; Y Chromosome