Effect of Extracts from Veronica peregrine on Breast Cancer Cell-induced Osteoclastic Bone Metastasis via Inhibiting Runx2 Activation / 中国实验方剂学杂志

ObjectiveTo investigate inhibitory effect of extracts from Veronica peregrina （EVP） on the osteoclastic bone metastasis induced by breast cancer cells. MethodBone metastasis model was established by injection of MDA-MB-231 cells， a human breast cancer cell line， into the left ventricle of BALB/c nude mice. The expression of human cytokeratin-19 （Ck-19） gene in mouse bone marrow was determined by nested polymerase chain reaction（PCR） to assess the bone metastasis of MDA-MB-231 cells. To assess the effects of EVP on the activation of bone marrow macrophages （BMMs）， we counted the multinuclear cells and measured the secretion of Cathepsin K. Western blot was adopted to assess the effects of EVP on receptor activator of nuclear factor-κB （RANK）， Runt-related transcription factor 2 （ Runx2 ）， phosphorylated Runx2 （p-Runx2）， and matrix metalloproteinase-9 （MMP-9） in BMMs. Gelatin zymography was employed to determine the activities of matrix metalloproteinases （MMPs）. ResultCompared with that in the blank group， Ck-19 expression was down-regulated in EVP groups （P<0.05）. The multinucleated cells increased when the BMMs were induced by soluble receptor activator of nuclear factor-κB ligand （sRANKL）， which was inhibited by EVP （P<0.05）. The level of cathepsin K in the supernatant of sRANKL group increased compared with that of the blank group， while EVP groups had lower cathepsin K levels than sRANKL group （P<0.05）. Compared with the blank group， the sRANKL group showed up-regulated RANK expression， Runx2 phosphorylation， and MMP-9 expression （P<0.05）， while the expression levels of RANK， p-Runx2， and MMP-9 were down-regulated when the cells were incubated with EVP （P<0.05）. Furthermore， exposure of BMMs to sRANKL resulted in an increase in gelatin hydrolyzation compared with the blank group （P<0.01）， which， however， was reversed in EVP groups （P<0.05）. ConclusionEVP significantly inhibits bone marrow metastasis of MDA-MB-231 cells， which may be associated with the suppression of osteoclast activation by inhibiting Runx2 phosphorylation.

Danshen Injection Inhibits SKOV3 Cell Proliferation in Vitro by Interfering with Their Interaction with Platelets / 中国实验方剂学杂志

Objective:To investigate the inhibitory effects of Danshen injection against ovarian cancer cell proliferation induced by the interaction between platelets and cancer cells. Method:The induction of platelets on SKOV3 growth <italic>in vitro</italic> and the inhibitory effect of Danshen injection at 12，24，and 48 g·L^-1 were observed by 3-（4，5-Dimethylthiazol-2-yl）-2，5-diphenyltetrazolium bromide （MTT） and cell colony formation assays. The content of transforming growth factor-<italic>β</italic>₁ （TGF-<italic>β</italic>₁） in the platelet-tumor cell interaction system and platelet supernatant and the effect of Danshen injection on TGF-<italic>β</italic>₁secretion were detected by enzyme-linked immunosorbent assay （ELISA）. The influences of tumor cell culture supernatant on platelet aggregation and secretion and the inhibitory effect of Danshen injection were determined by microplate assay and ELISA. The effects of Danshen injection on platelet nuclear factor kappa B （NF-<italic>κ</italic>B） signaling pathway were assayed by Western Blot. Result:Compared with the blank group， the platelet induction group exhibited significantly elevated absorbance at <italic>A</italic>₅₇₀（<italic>P</italic><0.01）， while the absorbance at <italic>A</italic>₅₇₀ in the platelet + Danshen injection group was significantly lower than that in the platelet induction group （<italic>P</italic><0.01）. The comparison with the Danshen injection group revealed that the cell proliferation inhibitory rate in the platelet + Danshen injection group at the same dose was more significant （<italic>P</italic><0.01）. The number of colonies in the platelet induction group was obviously increased in contrast to that in the blank group（<italic>P</italic><0.05）， while the number of colonies in the platelet + Danshen injection group was significantly lower than that in the platelet induction group（<italic>P</italic><0.05，<italic>P</italic><0.01）. As demonstrated by comparison with the blank group， TGF-<italic>β</italic>₁content in the supernatant of the platelet induction group rose remarkably（<italic>P</italic><0.01）， whereas that in the platelet + Danshen injection group declined（<italic>P</italic><0.01）. Compared with the Danshen injection （24 g·L^-1） group， the platelet + Danshen injection group displayed more obvious inhibition（<italic>P</italic><0.01）. Compared with the blank group， Danshen injection significantly reduced the TGF-<italic>β</italic>₁content in platelet supernatant（<italic>P</italic><0.05，<italic>P</italic><0.01）. There was no significant change in the content of TGF-<italic>β</italic>₁in SKOV3 supernatant treated with Danshen injection. The platelet aggregation， thromboxane A₂（TXB₂）， and serotonin （5-HT） secretion in the SKOV3 cell supernatant induction group were significantly increased as compared with those in the blank group （<italic>P</italic><0.01）， while such indexes in the cell supernatant induction + Danshen injection group were obviously decreased （<italic>P</italic><0.01）. Compared with the Danshen injection （24 g·L^-1） group， the cell supernatant induction + Danshen injection group displayed more obvious inhibition at the same dose（<italic>P</italic><0.01）. Compared with the blank group， the platelet induction group exhibited obviously up-regulated phosphorylated TGF-<italic>β</italic>-activated kinase-1 （TAK-1） and NF-<italic>κ</italic>B， but down-regulated phosphorylated inhibitory protein of NF-<italic>κ</italic>B （I<italic>κ</italic>B）（<italic>P</italic><0.01）， which however were significantly reversed in the platelet + Danshen injection group<bold>（</bold><italic>P</italic><0.01）. Conclusion:Danshen injection affect the proliferation of SKOV3 cells by inhibiting their interaction with platelets， which may be related to the inhibited secretion of TGF-<italic>β</italic>₁.

Mechanism of Duhuo Jisheng decotion in delaying degeneration of nucleus pulposus cells in human intervertebral disc / 中国中药杂志

This paper aimed to investigate the role of Duhuo Jisheng decotion (DHJSD) in delaying human disc degeneration and its possible molecular mechanism. The intervertebral disc specimens were divided into normal and degenerated groups according to Pfirrmann classfication. The expressions of TNF-α, IL-1β, MMP-3 and MMP-13 in intervertebral disc tissue were detected by Western blot and PCR. Then degenerated human primary NPCs were cultured in vitro, the viability of NPCs treated with stromal cell-derived factor-1 (SDF-1，10 μg·L⁻¹)and various concentrations of DHJSD was assessed by the CCK-8 assay, and the appropriate concentration was screened. The experiment was divided into three groups, control group, SDF-1 group and DHJSD plus SDF-1 group. The levels of TNF-α, IL-1β, Agg, coIⅡ, MMP-3 and MMP-13 were detected. The levels of CXCR4, NF-κB major groups P65 phosphorylation (p-P65) and nuclear translocation, after treated with CXCR4 siRNA and NF-κB inhibitor (BAY11-7082) were measured by Western blot and immunofluorescence. At the same time, the expression of cell inflammatory factors and extracellular matrix were also measured. The expressions of TNF-α, IL-1β, MMP-3 and MMP-13 in the degenerated intervertebral disc tissue were significantly increased. In vitro study, the results of CCK-8 indicated that the viability of NPCs was significantly increased when DHJSD concentration was 300 mg·L⁻¹. After the experiment was divided into three groups, compared with SDF-1 group, the expressions of TNF-α, IL-1β, MMP-3 and MMP-13 in DHJSD group were significantly decreased, but the expressions of Agg, coIⅡ were significantly increased. When CXCR4-siRNA was transfected into NPCs, SDF-1 increased expressions of CXCR4 and p-P65 and inhibited nuclear translocation of P65, whose effect was suppressed by CXCR4-siRNA and DHJSD. In addition, when BAY11-7082 was used to treat NPCs, the expression of TNF-α, IL-1β, MMP-3 and MMP-13 were significantly decreased. DHJSD could inhibit the production of inflammatory factors and promote the synthesis of extracellular matrix. The potential mechanism may be related to the SDF-1/CXCR4/NF-κB signaling pathway.

Comparison of methods for extraction of short RNA-DNA hybrids / 军事医学

Objective To compare the effects of three different methods for extracting short RNA-DNA hybrids, including the TRI reagent method , the phenol saturated with water method and the phenol saturated with Tris buffer method in order to facilitate studies on the biological function of RNA-DNA hybrids .Methods Short RNA fragments modifiedwith FAM at the 5′end and those modified with Cy 5 at the 5′end were synthesized .RNA and DNA fragments were annealed to form RNA-DNA hybrids.They were extracted with the above-mentioned 3 methods respectively .The extracted products were analyzed with electrophoresis .Results and Conclusion Short RNA-DNA hybrids can be extracted by the phenol saturated with water method and by the phenol saturated with Tris buffer method .The results can help study the function of short RNA-DNA hybrids .

Cloning,expression and biological activity of m6A binding protein YTHDF2 / 军事医学

Objective To construct the recombinant plasmid of YTH domain family 2(YTHDF2)and express it in E.coli in order to obtain YTHDF2 fusion protein that was capable of binding m 6A-modified RNA.Methods The coding region of YTHDF2 gene was amplified by RT-PCR.The recombinant plasmid pET-28a-YTHDF2 was constructed and expressed in E.coli.The fusion protein was purified by Ni2+-NTA resin affinity chromatography, while the fusion protein activity was analyzed by Ni2+-NTA magnetic spheres.Results and Conclusion The recombinant YTHDF2 protein was expressed in E.coli BL21(DE3)and purified.YTHDF2 fusion protein was capable of binding RNA with m 6A-modification. The preparation of YTHDF2 fusion protein provides an essential tool to study the biological function of RNA with m6A-modification.

Sesquiterpenes with anti-metastasis breast cancer activity from Chloranthus henryi / 中国中药杂志

To study sesquiterpenes with anti-metastasis breast cancer activity from Chloranthus henryi, ten sesquiterpenes ,zedoarofuran (1), chlorajapolide D (2), 4β, 8β-dihydroxy-5α(H)-eudesm-7(11)-en-8, 12-olide (3), curcolonol (4), lasianthuslactone A (5), chlomultin C (6), (1E,4Z)-8-hydroxy-6-oxogermacra-1(10), 4, 7(11) -trieno-12, 8-lactone (7), shizukanolide E (8) , shizukanolide F (9) , 9α-hydroxycurcolonol (10), and five bis-sesquiterpenes, shizukaol B (11), shizukaol C (12) , cycloshizukaol A (13) , sarcandrolide B (14) , henriol A(15), were isolated by using different kinds of column chromatography methods from the ethyl acetate part of Ch.henryi and their structures were identified based on spectroscopic methods. Compounds 2, 8, 9, and 10 were obtained from the genus Chloranthus for the first time. Compounds 2, 5, 8-10, 12,and 14 were obtained from this plant for the first time. Some isolated compounds were subjected to evaluate the anti-metastasis breast cancer activity by using pharmacological methods, and only compounds 4, 11, and 12 were potent active.

Study on the ELISA Double-antibody Sandwich Method and Pharmacokinetics of Recombinant Human Calcineurin B Subunit in Rat Plasma / 中国药房

OBJECTIVE:To establish a method for determining recombinant human calmodulin B subunit(rhCNB)in rat plas-ma,and study its pharmacokinetics characteristics. METHODS:ELISA double-antibody sandwich method was adopted. 1 μg/ml rhCNB monoclonal antibody mAb was wrapped,added to the to-be-test sample,rhCNB polyclonal antibody pAb(dilution ratio of 1∶5 000)and HRP-labeled conjugate of anti-IgG(dilution ratio of 1∶10 000)were added. Using tetramethylbenzidine for develop-ing,microplate reader was conducted in wavelength of 450 nm to determine the absorbance value(OD value)and plasma concen-tration of 6 rats after 2,15,30,60,120,240,480,720 min of iv 2.5 mg/kg rhCNB,and the pharmacokinetic parameters were calculated by BAPP 3.0 software. RESULTS:The linear range of rhCNB were 0.195-12.5 ng/ml(r2=0.995 0),lower limit of quan-titation was 0.195 ng/ml,accuracy were 97.300%-103.622%(RSD<7.5%,n=6);RSDs of within-batch,inter-batch,freezing and thawing 3 times were no higher than 8.5%(n=6,18,15). rhCNB pharmacokinetics characteristics in rat fitted to two-com-partment model,AUC0-720 min was 173.038 mg·min/L and t1/2 was 94.62 min. CONCLUSIONS:The established method has high specificity and sensitivity,good accuracy and precision,which can be used for rhCNB quantitative detection and pharmacokinetics study in biological samples.

Analysis of projects funded by NSFC in field of pharmacology of traditional Chinese medicine between 2010 to 2013 fiscal years / 中国中药杂志

Projects which supported by National Natural Science Foundation of China (NSFC) in discipline of pharmacology of Chinese medicine between 2010 to 2013 financial years were reviewed. Based on these research items, new features and problems were summarized in this field.

Chemical constituents from seeds of Brassica campestris / 中国中药杂志

Fourteen compounds were isolated by column chromatography from the ethyl acetate extract of the seeds of Brassica campestris. Their structures were elucidated by physicochemical properties and spectroscopic data analysis. The isolated compounds were respectively identified as (5Z,7E)-4, 4-dimethyl-5-acetyl-5, 7-nonadienoic acid (1), indole-3-carboxaldehyde (2), blumenol A (3), vinylsyringol (4), sinapinic acid (5), sinapic acid ethyl ester (6), protocatechuic acid (7), crinosterol (8), campesterol (9), 7-oxo-stigmasterol (10), kaempferol (11), 2,5-dihydroxybenzoic acid (12), syringic acid (13) and daucosterol (14). Compound 1 was a new compound and the other compounds were isolated from this plant for the first time except for compounds 4, 5 and 13.

Effects of Jinhuang Yidan Granule on the bile compositions of primary bile duct pigment calculus patients / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To study the effects of Jinhuang Yidan Granule (JYD) on the bile compositions of primary bile duct pigment calculus patients.</p><p><b>METHODS</b>Sixty-six patients with primary bile duct pigment calculus were randomly assigned to the control group (who took no Chinese medicine) and the JYD group (who took JYD). The bile from T-tube during the operation, 3, 10, and 40 days after medication were examined. The contents of bile acids, bilirubin (conjugated bilirubin, mono-conjugated bilirubin), glucoprotein, calcium ion, beta-glucuronidase, superoxide radical anion, and other components were detected and compared.</p><p><b>RESULTS</b>Three days after taking JYD, the total bile acids increased, the total bilirubin and beta-glucuronidase decreased, showing statistical significance when compared with the control group (P < 0.05). In the JYD group, the total bile acid increased, the total bilirubin, the conjugated bilirubin, the mono-conjugated bilirubin, glucoprotein, calcium ion, beta-glucuronidase, superoxide radical anions decreased 10 and 40 days after medication, showing statistical significance when compared with the control group (P < 0.05, P < 0.01). The level of the total bile acid increased, the levels of the total bilirubin, the conjugated bilirubin, the mono-conjugated bilirubin, glucoprotein, calcium ion, beta-glucuronidase, superoxide radical anions decreased after 40-day medication in the two groups, showing statistical significance when compared with the peri-operative indices of the same group (P < 0 05, P < 0.01).</p><p><b>CONCLUSIONS</b>JYD could significantly improve the pathologic bile compositions of the bile duct calculus, improve the environment of the biliary tract, showing certain preventive and therapeutic effects on bile pigment calculus of the primary bile duct calculus. Better effects may be obtained by long-term taking.</p>

Study on the relationship between the serum levels of Th1/Th2 cytokines and the clinical manifestations of chronic hepatitis C and the outcome of interferon therapy / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigated the relationship between the serum levels of Th1/Th2 cytokines and the progress of viral hepatitis C and the outcome of interferon therapy.</p><p><b>METHODS</b>Serum cytokine detection used the method of ELISA. HCV genotype were classified by direct sequencing. HCV RNA loads were determined by fluorescence quantitative PCR.</p><p><b>RESULTS</b>The levels of IL-2 and TGF-beta in serum of patients with chronic hepatitis C were lower hut IL-5 was higher than those of normal control. The level of IL-6 was positively related to the sera level of ALT and was negatively related to sera HCV RNA load. Patients of HCV genotype 1 had higher sera quantities of IL-6 than those of genotype 2 and patients of genotype 2a had lower sera quantities of IL-2 than those of 2b. The levels of IL-2 had the tendency to decrease whereas IL-6 had the tendency to increase when time went on. The level of TGF-beta increased at early phase but decrease later. There were no difference of all cytokines detected between the groups of response and nonresponse before interferon therapy, hut the quantity of serum IFN-gamma were increased after interferon therapy in the response group.</p><p><b>CONCLUSION</b>The tested cytokines co-participate in the pathogenesis of chronic hepatitis C and have the relationship with the clinical manifestations of the patients. There were no correlation between the levels of Th1/Th2 cytokines in the serum before IFN treatment and the result of IFN therapy. Increasing IFN-gamma in the serum induced by IFN treatment is associated with sustained virological response.</p>

Contribution of co-contraction to arm impedance in the free planar movement / 医用生物力学

Objective Impedance control plays an important role in stability.This paper intends to explore such mechanism through modeling human reaching movement.Method Implemented with revised model,we ap-ply optimal control theory to neuro-muscle-skeleton model to calculate the stiffness ellipses.Result Com-pared with the original model and experimental figures,the model we proposed could overcome the shortage of monotonous changing of the original one and fit the data better.Conclusions So that this paper concludes that co-contraction contributes to impedance control even during free upper limb planar movement.

Contribution of co-contraction to arm impedance in the free planar movement / 医用生物力学

Objective Impedance control plays an important role in stability.This paper intends to explore such mechanism through modeling human reaching movement.Method Implemented with revised model,we ap-ply optimal control theory to neuro-muscle-skeleton model to calculate the stiffness ellipses.Result Com-pared with the original model and experimental figures,the model we proposed could overcome the shortage of monotonous changing of the original one and fit the data better.Conclusions So that this paper concludes that co-contraction contributes to impedance control even during free upper limb planar movement.

Reconstruction of large limb bone defects with a double-barrel free vascularized fibular graft / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The use of a free, vascularized fibular graft is an important technique for the reconstruction of large defects in long bones. The technique has many advantages in strong, tubular bones; a more reliable vascular anatomy with a large vascular diameter and long pedicle is used, minimizing donor-site morbidity. Due to limitations in both fibular anatomy and mechanics, they cannot effectively be used to treat large limb bone defects due to their volume and strength.</p><p><b>METHODS</b>From 1990 to 2001, 16 clinical cases of large bone defects were treated using vascularized double-barrel fibular grafts. Patients were evaluated for an average of 10 months after surgery.</p><p><b>RESULTS</b>All the patients achieved bony union; the average bone union took 10 months post surgery, and no stress fractures occurred. Compared with single fibular grafts, the vascularized double-barrel fibular grafts greatly facilitate bony union and are associated with fewer complications, suggesting that the vascularized double-barrel fibular graft is a valuable procedure for the correction of large bone defects in large, long bones in addition to enhancing bone intensity.</p><p><b>CONCLUSIONS</b>The vascularized double-barrel fibular graft is superior to the single fibular graft in stimulating osteogenous activity and biological mechanics for the correction of very large bone defects in large, long bones. Free vascularized folded double-barrel fibular grafts can not only fill up large bone defects, but also improve the intensity margin. Therefore, this study also widens its application and enlarges the treatment targets. However, in the case of bone deformability, special attention should be paid to bone fixation and protection of donor and recipient sites.</p>

External fixation and bone grafting for collapsed and comminuted distal radius fracture / 中华创伤杂志(英文版)

<p><b>OBJECTIVE</b>To study the therapeutic effect of collapsed and comminuted distal radius fracture.</p><p><b>METHODS</b>Twenty-six patients with collapsed and comminuted distal radius fracture were hospitalized from July 1998 to June 2003. All fractures were treated by the methods of open reduction, sustained bone grafting and passing joint external fixator to restore the anatomic shape of distal radius.</p><p><b>RESULTS</b>All 26 cases were followed up, and the results showed that the fractures have been united radiographically. The joint surfaces were intact and there was no length discrepancy occurred in patient's radius. The average volar tilt was 6 to 15 degrees and the average ulnar tilt was 18 to 25 degrees. According to the Dieust criterion, 19 cases were rated as excellent and 7 as good.</p><p><b>CONCLUSIONS</b>The method that applying passing joint external fixator and bone grafting for the treatment of collapsed and comminuted distal radius fracture could maintain the stability of fracture and restore the length of radius and the intact of joint surface.</p>

Detection of a new mutation (G1253T) of iduronate-2-sulfatase gene for the patient with mucopolysaccharidosis type II / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To identify the mutations of iduronate-2-sulfatase (IDS) gene in mucopolysaccharidosis type II patients.</p><p><b>METHODS</b>PCR-SSCP analysis was applied to detect the common mutations in the exons 2, 3, 5, 7, 8, 9 in IDS-gene of the patient. DNA sequencing and PCR-RFLP were applied to analyze the mutation detected by PCR-SSCP.</p><p><b>RESULTS</b>A new mutation(1253G-->T) of exon 7 of the IDS gene was found by PCR-SSCP and DNA sequencing in the patient, The PCR-restriction enzyme digestion showed that enzyme digestion location appeared in the patient and his mother, which verified the results of sequencing analysis.</p><p><b>CONCLUSION</b>The mutation of patient with MPSII could be detected effectively and quickly by the applications of PCR-SSCP, DNA sequencing and PCR-restriction enzyme digestion analysis, and the new mutation thus detected is necessary for the prenatal diagnosis of the pedigree.</p>

Cloning and expression of hTRF1 in Escherichia coli and preparation of polyclonal antibody / 生物工程学报

Human telomeric repeat binding factor 1(TRF1) contains one Myb-type DNA-binding repeat and an amino-terminal acidic domain. It can bind to the duplex array of TTAGGG repeats at chromosome ends and is shown to be important in preserving genomic stability, maintaining cell proliferative capacity, and blocking the activation of DNA-damage cell cycle checkpoints. Interestingly, the double strand DNA breaks sensor ATM interacts with and phosphorylates Pin2/TRF1 and inhibits its function after DNA damage. Are there some proteins else that can interact with TRF1 and influence its function? In order to analysis the interaction between TRF1 and other proteins, we must prepare the antiserum that can recognize the endogenous TRF1 of cell lysates. TRF1 cDNA was amplified using cDNA Library of HeLa cell by PCR and cloned into pUCm-T vector. Sequence analysis reveals identity to the GenBank report. The TRF1 cDNA was subcloned into expression vector pET-28c(+) and expressed in E. coli as a fusion protein of 65 kD. The recombinant TRF1 can express in the supernatant (about 12.3% in total protein) on the induction of 0.5 mmol/L IPTG at 37 degrees C for 3 hours. Western-blot analysis showed the recombinant protein can react with TRF1 polyclonal antibody sc-6165 (from Santa Cruz Company). His6-TRF1 was purified by Ni(2+) -NTA resin affinity chromatography made by ourselves and showed to be homogeneity in SDS-PAGE. Rabbits were immunized for four times to prepare polyclonal antibody. The unpurified antiserum can recognize the overexpressed TRF1 with myc-tag and the endogenous Pin2/TRF1 of cell lysate by Western-blot at 1:1000 dilution. At 1:400 dilution, the antiserum can interact with endogenous TRF1 by Immunofluorescence cell staining analysis. The endogenous TRF1 in different cell lines, such as HepG2, 803, MCF7 and HeLa, locates in the nucleus. The soluble expression TRF1 and preparation of its antibody lay the foundation to study it further.

46,XY female sex reversal patient with a novel point mutation in the coding sequence of the SRY gene / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the molecular mechanism of a Chinese patient with 46, XY sex reversal.</p><p><b>METHODS</b>DNA fragments of the SRY gene from the typical XY female sex reversal patient and her father were amplified by polymerase chain reaction (PCR). The amplified PCR fragments were cloned into the pUCm-T vector, and direct sequencing was carried out on an ABI 377-3 automated DNA sequencer to detect the mutation. PCR-restriction enzyme digestion was applied to detect the results of DNA sequencing.</p><p><b>RESULTS</b>A novel mutation of the SRY gene was identified in the XY sex reversal patient of this study. A T is replaced by an A in codon 129 at position +387, resulting in the replacement of the polar amino acid tyrosine (TAT) by the stop code (TAA) in the HMG-box, whereas her father was proved to have the wild-type sequence. Because the mutation introduced an enzyme site of MaeIII, the PCR-restrict enzyme digestion showed that there were three bands (131 bp,231 bp and 247 bp) in the patient, whereas two bands (131 bp and 478 bp) in normal man. It verified the results of sequencing analysis. The results after searching the Human Gene Mutation Database showed that this mutation was not described before and should be a new mutation.</p><p><b>CONCLUSION</b>The novel mutation in SRY gene has provided valuable information for the understanding of molecular mechanism of the patient with 46,XY female sex reversal.</p>

Diagnosing achondroplasia by single cell nested-PCR / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To research on the reliability of diagnosing achondroplasia (ACH) on single cell level and to provide a basis for preimplantation genetic diagnosis(PGD).</p><p><b>METHODS</b>The high-frequency mutation region G380R of fibroblast growth factor receptor 3(FGFR3) gene was amplified by nested-PCR with single lymphocyte and single blastomere. The products of PCR were digested by restriction enzyme Bfm I, then the digested products were detected by 10% polyacrylamida gel electrophoresis(PAGE).</p><p><b>RESULTS</b>The amplification success rate, allele dropout rate and correct diagnosis rate of single lymphocyte's PCR were 90.4%, 8.2% and 91.8%,respectively. The amplification success rate of single blastomere was 75.4%.</p><p><b>CONCLUSION</b>The diagnosis of ACH by single cell nested-PCR is comparatively stable and reliable.</p>

Inducing vascular smooth muscle cell proliferation by insulin involves in phosphatidylinositol 3-kinase and ERK1/2 / 中国病理生理杂志

AIM:To investigate the effect of insulin on vascular smooth muscle cells (VSMCs) proliferation and to evaluate the intracellular signaling pathways involved.METHODS:VSMCs separated from Sprague-Dawley rats were used in this study. The proliferation of VSMCs induced by insulin was assayed by [3H]-thymidin incorporation. The protein expression and activity of p-ERK1/2 were determined by immunblot and [?-32P]ATP incorporation.RESULTS:Insulin induced cell proliferation in a concentration-dependent manner. The proliferative effect of insulin on VSMCs was inhibited partly by LY294002 (48.8%),an inhibitor of PI-3 kinase,and the ERK1/2 inhibitor PD98059 (43.6%),respectively. Moreover,phosphorylation of ERK1/2 and activity of ERK1/2 induced by insulin were also inhibited partly by LY294002.CONCLUSION:PI-3 kinase and ERK1/2 are involved in insulin induced VSMCs proliferation.