OBJECTIVE: To observe the effects of rhubarb aglycone on pathological changes and activity of matrix metalloproteinase in cerebral ischemic tissue in rats with bone marrow mesenchymal stem cell (BMSC) transplantation, and to explore the action mechanisms of rhubarb aglycone in protecting against brain micrangium injury in rats. METHODS: The BMSCs were purified and amplified by methods of adherence and selection in vitro. One hundred and ninety rats were randomly divided into sham-operated group, untreated group, rhubarb aglycone group, BMSC transplantation group (abbreviated as transplantation group) and BMSCs combined with rhubarb aglycone group (abbreviated as combination group). Middle cerebral artery occlusion (MCAO) model was duplicated with nylon thread. Rats of transplantation and combination group were transplanted with BMSCs via carotid artery after 24-hour reperfusion. Rhubarb aglycone was used by intragastric administration in the rhubarb aglycone group and the combination group. The brain samples were taken at 7, 14 and 28 days after transplantation. Brain micrangium pathological changes were observed by light microscope, and immunohistochemical method was used to determine the expressions of immunoglobulin G (IgG), type IV collagen (Col IV), matrix metalloproteinase-9 (MMP-9), and tissue inhibitor of metalloproteinase-1 (TIMP-1). RESULTS: Comparison with the normal control group revealed that brain micrangium in rats in the untreated group was obviously mutilated and damaged, the expression of IgG and MMP-9 increased, and showed a progressively enhanced tendency following the prolongation of reperfusion, while the expressions of Col IV and TIMP-1 were decreased, and TIMP-1 showed a attenuated tendency following the prolongation of reperfusion. Comparing with the untreated group, the improvements of brain micrangium structure in the rhubarb aglycone group (7 days after transplantation), the transplantation group (14 and 28 days after transplantation) and the combination group were significant; expression of IgG and activity of MMP-9 were decreased, while expressions of Col IV and TIMP-1 were increased in the rhubarb aglycone group and the combination group at each time point. The brain micrangium was integral and the expression of Col IV was enhanced in combination group (7 days after transplantation) as compared with those in transplantation group. MMP-9 activity in combination group (14 days after transplantation) was lower than that in the rhubarb aglycone group (14 days after transplantation), while expression of TIMP-1 in combination group also increased significantly as compared with that in transplantation group (28 days after transplantation). CONCLUSION: Rhubarb aglycone can decrease the degradation of basal lamina Col IV and the permeability of brain micrangium in cerebral ischemic rats with BMSC transplantation, and its mechanisms may be related to regulating the balance of MMP-9, especially by increasing the expression of TIMP-1.

OBJECTIVE: To study the protective effects of Naomaitong, a compound traditional Chinese herbal medicine, used together with thrombolysis therapy, on injuries of the lung and stomach in rats with cerebral ischemia. METHODS: Rats were randomly divided into sham-operated group, untreated group, urokinase group (thrombolysis group), Naomaitong group, thrombolysis plus Naomaitong group. Cerebral ischemia was induced in rats by autonomous blood blot and inserted nylon thread. Rats were administrated with thrombolysis therapy through artery 3, 6 and 9 h after cerebral ischemia respectively. Twenty-four hours after the administration, mortality of the rats, and the brain and stomach hemorrhage ratios, as well as the pathological changes of the brain, lung and stomach were observed, and then cerebral infarct size (CIS) and lung water ratio (LWR) were measured. RESULTS: Compared with the sham-operated group, the rat mortality, and the brain and stomach hemorrhage ratios increased, the CIS enlarged, pathological changes of the brain, lung and stomach appeared obvious, and the LWR increased. Naomaitong plus thrombolysis treatment improved the changes above significantly. In the untreated rats with cerebral ischemia, injuries of the brain, lung and stomach were aggravated, the CIS enlarged and the LWR increased in the 9 h group as compared with those in the 3 h group. In the thrombolysis plus Naomaitong group, the pathological changes were improved, the CIS diminished and the LWR reduced. CONCLUSIONS: Injuries of the lung and stomach can be caused by cerebral ischemia, and the impairment was exacerbated following the prolongation of the ischemia. Thrombolysis therapy can cause brain and stomach hemorrhage. Thrombolysis therapy used early can perform protection against the injuries. Naomaitong, used together with thrombolysis, can reduce the mortality, the brain and stomach hemorrhage ratios, and perform protective effects on the injuries of cerebral ischemia.

The glutamate dehydrogenase (EC.1.4.1.4) gene which amplified from the genome of Brevibacterium flavum GDK-9 by polymerase chain reaction was linked with pUCm-T for sequence alignment. Analysis of gdh sequences revealed that the whole sequence is 1927 bp, only one ORF existed, which used ATG as the initiation codon and coded a peptide of 448 amino acids with a calculated molecular weight of 48 kD. The comparability between the cloned gdh sequence to the reported sequence is high to 99.55%. Only the 1190th base mutation (C→A) lead to the change of amino acid sequence (Thr→Asn), the others are not. The recombinant plasmid pXG was then transformed into E. coli XL-Blue and Brevibacterium flavum GDK-9 which was induced by IPTG. SDS-PAGE analysis revealed that there was a clear induced protein band with molecular mass of 48.7 kD on expected position. Standard glutamate fermentations indicated that although the level of GDH increases the intracellular glutamate pool, the level of GDH has no influence on glutamate secretion.

[Summary] All patients with cholesterol side-chain cleavage enzyme ( P450scc) deficiency that have been reported presented with early adrenal failure. Here we described a 35-year-old male presented with infertility as the only initial presenting complaint. He had received two separate surgeries to remove bilateral testicular masses. We reevaluated the resected tumors and found testicular adrenal rest tumor ( TART) pathology in the resected tumor. We profiled steroid hormones and found significantly elevated ACTH. CT scan revealed bilateral adrenal hyperplasia. Mutation screening identified compound heterozygous mutations (R353W and P432L) in the P450scc encoding gene (CYP11A1). The patient was finally diagnosed as congenital adrenal hyperplasia.

Objective To evaluate the value of endobronchial ultrasound elastography in diagnosis of mediastinal lymph nodes by measuring the parameters of endobronchial ultrasound elastography. Methods Between 2016 and 2017, 46 patients with 63 lesions underwent EBUS-TBNA examination were included. Conventional ultrasound and ultrasound elastography were performed respectively to examine 63 lymph nodes before aspiration. Then various features of conventional ultrasound and parameters of ultrasound elastography were analyzed and recorded. Then evaluate the value of ultrasound elastography by pathological results and three-month follow-up data. Result The diagnostic value of ultrasound elastography image type, elasticity score, strain ratio and blue area ratio for malignant lymph nodes were higher than that of conventional ultrasound features in accuracy, sensitivity and specificity (P < 0.05); The accuracy of the boundary was the highest in the diagnosis of malignant lymph nodes. In the ultrasound elastography parameters, the accuracy of the elasticity score was the highest, and the blue area ratio had the largest area under the curve. When the boundary was used as the joint index to judge malignant lymph nodes with elasticity score and area ratio respectively, significantly higher than the diagnostic accuracy of conventional ultrasound and ultrasound elastography alone (P < 0.05). Conclusion The value of ultrasound elastography parameter in mediastinal lymph nodes diagnosis is higher than the conventional ultrasound features and a combination of both applications can improve discrimination accuracy of benign and malignant lymph nodes.

Objective To assess the association of synovial cyclic citrullinated peptide(CCP)expression with T helper 17(Th17) cells/Regulatory T cells (Treg) imbalance,the histological and clinical features of synovitis in rheumatoid arthritis (RA).Methods CCP expression in synovial specimens from 39 patients with RA and 35 controls was detected by immunohistochemistry assay(IH) using 6×His tagged anti-CCP single chain fragment V (ScFv) antibodies,which were generated by pHEN2 phagemid recombinant antibodies display system.The frequencies of Th17/Treg cells in the peripheral blood mononuclear cells (PBMCs) were determined by flow cytometry (FCM).Th17/Treg cells associated cytokines were analyzed by enzyme-linked immunosorbent assay (ELISA).The histological scores and clinical features of synovitis were included in the study.Chi-square test and ANOVA were used for statistical analysis.Results ① The prevalences of synovial C CP expression were significantly different between RA group and the control(76.9％ and 11.4％ respectively,X2=31.9,P＜0.01).② The frequencies of Th17 cells,Th17/Treg ratio,Th17 cells associated cytokines as IL-6,IL-17a,IL-23,TNF-α,and the Treg cells associated cytokines TGF-31,the serum and synovial fluid anti-CCP antibodies in the RA patients with synovial CCP positive expression were significantly higher in RA patients with CCP positive than those with CCP negative.Disease activity score DAS28 index and histological features quantified variations of the synovial biopsy specimens (synoviocyte hyperplasia,focal aggregates of lymphocytes,and diffuse infiltrat(e)s of lymphocytes) in RA were higher in synovial CCP positive expression patients than in the negative.Conclusion Synovial CCP expression is strongly associated with the Th17/Treg imbalance and synovitis,which may play a crucial role in the pathogenesis of RA.

OBJECTIVETo construct the expression vectors of vWF73 and vWF114 fragments of von Willebrand factor (vWF) A2 domain, and to express glutathione S-transferase (GST) fusion proteins in E. coli, and to explore their values in measuring ADAMTS13 activity as substrates.

METHODSThe DNA fragments encoding vWF73 and vWF114 were generated using PCR and separately cloned into pGEX-6P-1, a Schistosoma japonicum GST fusion expression vector. The expression of GST-vWF73-H and GST-vWF114-H was induced in liquid culture, followed by purification with Ni-NTA agarose column. The cleavage of two GST fusion proteins by recombinant ADAMTS13 (rADAMTS13) or plasma from normal individuals and thrombotic thrombocytopenic purpura (TTP) patients were identified by Western blot. Based on an enzyme-linked immunosorbent assay (ELISA) with anti-GST and anti-His monoclonal antibodies, GST-vWF73-H and GST-vWF114-H were used to measure plasma ADAMTS13 activity as substrates.

RESULTSTwo small molecular substrates of ADAMTS13, GST-vWF73-H and GST-vWF114-H, are expressed and purified, which could be specifically cleaved by rADAMTS13 or plasma from healthy individuals, but not by plasma from congenital or idiopathic TTP patients. An ELISA assay was established to detect plasma ADAMTS13 activity using GST-vWF73-H and GST-vWF114-H as substrates.

CONCLUSIONSTwo GST fusion proteins in vWF A2 domain, vWF73 and vWF114, were expressed effectively using a prokaryotic expression system and could be used to detect ADAMTS13 activity as substrates.

ADAM Proteins ; blood ; genetics ; ADAMTS13 Protein ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; metabolism ; Glutathione Transferase ; metabolism ; Humans ; Male ; Purpura, Thrombotic Thrombocytopenic ; blood ; genetics ; metabolism ; von Willebrand Factor ; genetics ; metabolism

OBJECTIVETo investigate the susceptibility of von Willebrand factor (VWF) type 2A mutant A1500E to proteolysis by metalloprotease ADAMTS13 and to provide the direct supports for the pathogenesis of VWF mutation A1500E responsible for von Willebrand disease (VWD) type 2A.

METHODSRecombinant wild-type VWF (WT-VWF) and A1500E mutant VWF transiently expressed on transfected HeLa cell lines. Expression media were collected and concentrated, then cleaved directly by recombinant ADAMTS13 (rADAMTS13). Compared with WT-VWF, the susceptibility of A1500E mutant VWF to proteolysis by ADAMTS13 was analyzed using SDS-agarose gel VWF multimers analysis.

RESULTSIn vitro the expression of VWF:Ag in the supernatants of WT-VWF and A1500E mutant VWF were 1.10 U/ml and 0.78 U/ml, respectively, while VWF:Ag in cells lysates of A1500E mutant VWF was 90.6% of that of WT-VWF. The SDS-agarose gel VWF multimers analysis showed that there were no differences between WT-VWF and A1500E mutant VWF. The A1500E mutant VWF could be efficiently cleaved by ADAMTS13 under static condition without denaturants such as urea and guanidine HCl. VWF multimeric analysis showed that high and intermediate molecular weight multimers dramatically decreased while low molecular weight multimers obviously increased. Conversely, WT-VWF could not be cleaved by ADAMTS13 under the same condition.

CONCLUSIONThe A1500E mutation resulted in VWF more susceptible to ADAMTS13-dependent proteolysis, which belonged to VWD type 2A group 2 mutation.

ADAM Proteins ; genetics ; metabolism ; ADAMTS13 Protein ; Genotype ; HeLa Cells ; Humans ; Hydrolysis ; Mutation ; Recombinant Proteins ; genetics ; metabolism ; von Willebrand Disease, Type 2 ; genetics ; metabolism ; von Willebrand Factor ; genetics

OBJECTIVETo screen cellular proteins binding to the core region of hepatitis C virus (HCV) from human hepatoma cells.

METHODSUnlabeled and labeled RNA transcripts were prepared by in vitro transcription. Cytoplasmic extracts were prepared from human hepatoma cells HepG2. Ultraviolet (UV) cross-linking was used to screen the cellular proteins that would bind to the core region of HCV. Competition experiment was performed to confirm the specificity of the binding in which excess unlabeled RNA of HCV core region and plasmid RNA were used as competitors.

RESULTSTwo cellular proteins of 6.6 x 10(4) and 5.5 x 10(4) were found binding to the core region of HCV RNA by UV cross-linking assay. The unlabeled core region of HCV RNA could compete out this binding whereas the unlabeled plasmid RNA could not.

CONCLUSIONThe cellular proteins from HepG2 cells could bind to the core region of HCV RNA.

Binding Sites ; Cross-Linking Reagents ; chemistry ; Hepacivirus ; genetics ; metabolism ; RNA, Viral ; genetics ; metabolism ; Ultraviolet Rays ; Viral Core Proteins ; genetics ; metabolism

OBJECTIVETo make a comparatively analysis of the effects of 10% KCl injection into the fatal cardiac area and yolk-sac aspiration on multifetal pregnancy reduction.

METHODSTwenty-three patients with multifetus were selected in the investigation. Eight of the patients accepted 10% KCl injection into the fatal cardiac area, and 15 of them received yolk-sac aspiration. The average number of punctures, average time of reduction operation, failure rate of operation, abortion rate, and infection rate were observed.

RESULTSThe average time of reduction operation[(2.8 +/- 0.7) min] of aspiration was significantly lower than that of 10% KCl injection [(5.11 +/- 1.35) min] (P < 0.05). The infection rate of yolk-sac aspiration was 6.7%, lower than that of 10% KCl injection (37.5%) (P > 0.05). Cardic area injection showed a higher infection rate, and no significant difference was observed in the average number of punctures, failure rate of operation and abortion rate(P > 0.05).

CONCLUSIONAlthough both yolk-sac aspiration and cardiac area injection were safe and reliable methods for multifetal pregnancy reduction, the former is worth recommending for its shorter operation time heeded and lower infection rate, especially for the multifetal patients within 60 gestation days.

Adult ; Female ; Humans ; Postoperative Complications ; etiology ; Potassium Chloride ; administration & dosage ; Pregnancy ; Pregnancy Reduction, Multifetal ; adverse effects ; methods ; Ultrasonography, Prenatal