Objective To clone and express cathepsin B gene of Echinococcus granulosus(EgCatB)and analyze EgCatB protein by using bioinformatics tools and online databases. Methods The total RNA of E. granulosus was extracted and reverse?ly transcribed into cDNA as the template sequence for PCR. The EgCatB gene was cloned by using the In?Fusion PCR cloning method and expressed by a wheat germ cell?free system,and then the recombinant protein was identified by Western blotting. The signal peptide,transmembrane helices and subcellular location of the EgCatB sequence were predicted by the online soft?ware SignalP 4.1,TMHMM sever v. 2.0 and TargetP 1.1 respectively. Subsequently,the homologue sequence and conserved sites were aligned by using BLASTP and GeneDoc software. Finally,the structures and the glycosylation modification site of the EgCatB encoding protein were analyzed and predicted in turn by ProtParam,SMART,Predictprotein,Swiss?model,NetOGlyc 4.0 and NetNGlyc 1.0 approaches. Results The EgCatB gene was successfully amplified from cDNA of E. granulosus and ex?pressed in the soluble fractions. The molecular weight of the expressed protein was estimated 35 kDa. The bioinformatics analysis revealed that EgCatB was a classical secreted protein containing a Pept_C1 domain. The homology analysis indicated that the amino acid sequence of EgCatB was highly conserved in the active enzyme sites. The protein structure prediction showed a cata?lytic active center was formed through Gln106,Cys112,His282 and Asn302. It was found that there were nine O?glycosylation sites in the EgCatB sequence,but no N?glycosylation sites. Conclusions The EgCatB gene is cloned and expressed successfully,and the recombinant protein is analyzed by bioinformatics approaches and structure predication. The study provides useful informa? tion for further functional study of the EgCatB protein.

OBJECTIVETo study the effects of gonadotroph-releasing hormone (GnRH) agonist (GnRH-a) and GnRH antagonist (GnRH-ant) on cyclophosphamide (CTX)-induced follicle apoptosis in female rats.

METHODSThirty-six female Sprague- Dawley rats were randomized into 6 groups, namely normal saline (NS), CTX, GnRH-a+NS, GnRH-a+CTX, GnRH-ant+NS, and GnRH-ant+CTX groups. The rats were sacrificed between the first and second week after the treatments., and the follicle apoptosis was investigated using TUNEL assay and transmission electron microscopy.

RESULTSThe apoptosis rate of the granulose cells in the follicles in late development was significantly higher than that in early follicles, and the apoptosis rate of the oocytes and granulose cells in rats with CTX treatment was significantly higher than that in rats without CTX treatment (P<0.05). The apoptosis rate of the granulose cells in GnRH-a groups (ranging from 33.40 - or + 4.59 to 73.25 - or + 5.35) was significantly higher than that in GnRH-ant groups (27.46 - or + 4.52 to 49.38 - or + 5.02, P<0.05), but there was no significant difference in the oocytes of early follicles between GnRH-a groups (23.48 - or + 4.25 to 36.15 - or + 4.23) and GnRH-ant groups (21.47 - or + 3.81 to 34.04 - or + 5.54, P>0.05). Electron microscopy revealed characteristic apoptotic changes of the oocytes in early follicles and granulose cells in early and late follicles. The apoptotic changes were especially typical in the granulose cells showing the formation of the apoptotic bodies, and the oocytes only showed chromatin condensation and aggregation.

CONCLUSIONIn the rat mode, GnRH-a promotes while GnRH-ant suppressed follicle apoptosis induced by CTX. GnRH analogues regulates mainly granulose cell apoptosis, but have little effect on oocyte apoptosis.

Animals ; Apoptosis ; drug effects ; Cyclophosphamide ; toxicity ; Female ; Gonadotropin-Releasing Hormone ; analogs & derivatives ; antagonists & inhibitors ; Granulosa Cells ; pathology ; Oocytes ; pathology ; Ovarian Follicle ; pathology ; Random Allocation ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo study the regulattory effect of Astragalus membranaceus on immune disturbance of the rats with IgA nephropathy.

METHODSRats IgA nephropathy (IgAN) model was duplicated by oral feeding of bovine serum albumin (BSA), subcutaneous injection of carbon tetrachloride (CCl4) and injection of lipopolysaccharide (LSP) into vena caudalis. The rats were divided into three groups randomly for the normal, IgAN model group and the group treated with Astragalus membranaceus (treatment group). The treatment group was given the Astragalus membranaceus granules via intragastric administratsion, the normal group and the IgAN model group were given the equal amount of aqua destillata by gastric perfusion. The rats were examined for albuminuria, hematuria and pathological changes of renal tissue and the distribution of TGF-beta and interleukin-5 in renal tissue was determined by immunohistochemistry and the IFN-gamma and IL-4 of cytokine of Th1 and Th2 types were detected in rats IgA nephropathy model by sandwich enzyme linked immunosorbent assay (ELISA).

RESULTS(1) The hematuria in rats with IgA nephropathy significantly increased compared with normal control group and Astragalus treatment group (P < 0.05). There was significant increase in albuminuria in rats with IgA nephropathy, compared with normal control group and astragalus treatment group (P < 0.01). (2) The pathological change of glomerular mesangium, renal tubules and renal interstitia became serious in rats IgA nephropathy model when compared with normal control group and astragalus treatment group. Immumofluorescence showed renal IgA density in rats IgA nephropathy model was significantly higher than that in the normal control group (P < 0.001) and astragalus treatment group (P < 0.001). (3) The result of immuno histochemistry showed that there was only weak expression of TGF-beta and interleukin 5 in normal renal tissue. The expression of TGF-beta and interleukin 5 in IgA nephropathy model was significantly stronger than those in normal control group (P < 0.05) and astragalus treatment group (P < 0.05). (4) The serum IL-4 levels were (33.74 +/- 7.52) pg/ml in rats IgA nephropathy model, significantly higher than that in normal control group (2.36 +/- 0.85) pg/ml and astragalus treatment group (3.24 +/- 1.13) pg/ml. The IFN-gamma level in serum of rats IgA nephropathy model was (18.79 +/- 3.80) pg/ml, which was significantly higher than that in normal control group (46.53 +/- 5.56) pg/ml and astragalus treatment group (41.28 +/- 2.95) pg/ml.

CONCLUSIONSThe astragalus could lower the level of hematuria and 24 hours-albuminuria of the IgAN model, and amelioratse the change of the renal pathology and reduce the deposit of IgA in glomerular mesangium. The possible mechanism of the effect is that astragalus could regulate the derangement of Th1, Th2, accordingly could improve the level of IL-4 and IFN-gamma in the serum and diminish the expression of cytokine Th2 TGF-beta1 and IL-5 of the renal tissue, and thereby could postpone the development of IgAN.

Animals ; Astragalus membranaceus ; chemistry ; immunology ; Cattle ; Drugs, Chinese Herbal ; pharmacology ; Glomerulonephritis, IGA ; immunology ; Interleukin-4 ; pharmacology ; Interleukin-5 ; pharmacology ; Kidney Tubules ; drug effects ; Rats ; Transforming Growth Factor beta ; immunology ; Transforming Growth Factor beta1 ; pharmacology

OBJECTIVETo construct the siRNA plasmid for mfgl2 gene, which has been reported to be involved in a variety of disease developments including fulminant viral hepatitis, acute rejection of allo/zero transplantation and fetal loss syndrome, and to investigate its inhibitory effects on mfgl2 expression in vitro.

METHODSA plasmid p-mfgl2shRNA complimentary to the sequence responsible for the functional domain of mouse fgl2 (mfgl2) was constructed. The pcDNA3.1 mfgl2 expression construct was able to show a satisfactory fgl2 protein expression. The plasmid expression pEGFP and a construct expressing irrelevant shRNA with a random combination of the p-mfgl2shRNA sequence were used as controls. A pEGFP-mfgl2 expressing mfgl2-EGFP fusion protein was also constructed for screening of the effect of p-mfgl2shRNA on the mfgl2 expression.

RESULTSCotransfection of p-mfgl2shRNA with pEGFP-mfgl2 decreased green fluorescent cells and the lightness of fluorescence within the cells at the 24 h, 48 h and 72 h post-transfection when compared with that in the control groups which were solely transfected with pEGFP-mfgl2. Furthermore the mfgl2 expression was significantly reduced when the pcDNA3.1 mfgl2 expression construct was cotransfected with p-mfgl2shRNA both at mRNA level by RT-PCR and protein level by RT-PCR, immunohistochemistry staining and FACS in both CHO cell and Hela cell lines.

CONCLUSIONSThe study demonstrated that the construct of p-mfgl2shRNA successfully interfered in the mfgl2 expression in vitro. It provides a basis for a further investigation of effect in vivo.

Animals ; Fibrinogen ; biosynthesis ; genetics ; Gene Expression ; Mice ; Plasmids ; genetics ; RNA Interference ; RNA, Small Interfering ; genetics

OBJECTIVE: To evaluate the short and mid-term changes of the cardiac morphology after percutaneous transcatheter closure of ventricular septal defects (VSD) with transthoracic echocardiography (TTE). METHODS: The left ventricular end-diastolic diameter (LVEDD), left ventricular end-diastolic volume (LVEDV), left atrial diameter (LAd), and right ventricular diameter (RVd) in 30 VSD patients were measured before the VSD closure,and on the 3rd day, 3rd month, and 6th month after the VSD closure by TTE. RESULTS: LVEDD and LVEDV significantly decreased on the 3rd day after the VSD closure compared with pre-VSD closure. LVEDD and LVEDV continuously decreased on the 3rd month and 6th month after the VSD closure. LAd was smaller on the 3rd month and 6th month after the VSD closure, but there was not significant difference between the 3rd and 6th month. RVd increased on the 3rd day after the VSD closure, while no significant difference was found among the 3rd month and 6th month before and after VSD closure. CONCLUSION Percutaneous transcatheter VSD closure may effectively improve the cardiac remodeling in VSD patients in the short and mid-term follow-up.
Adolescent ; Adult ; Cardiac Catheterization ; methods ; Child ; Child, Preschool ; Echocardiography ; Female ; Follow-Up Studies ; Heart Septal Defects, Ventricular ; diagnostic imaging ; therapy ; Humans ; Male ; Prosthesis Implantation ; methods ; Time Factors ; Ventricular Remodeling

Background/Aims: Anti-reflux mucosal ablation (ARMA) is a promising endoscopic intervention for proton pump inhibitor (PPI)-dependent gastroesophageal reflux disease (GERD). However, the effect of ARMA on esophageal motility remains unclear. Methods: Twenty patients with PPI-dependent GERD receiving ARMA were prospectively enrolled. Comprehensive self-report symptom questionnaires, endoscopy, 24-hour impedance-pH monitoring, and high-resolution impedance manometry were performed and analyzed before and 3 months after ARMA. Results: All ARMA procedures were performed successfully. Symptom scores, including GerdQ (11.16 ± 2.67 to 9.11 ± 2.64, P = 0.026) and reflux symptom index (11.63 ± 5.62 to 6.11 ± 3.86, P = 0.001), improved significantly, while 13 patients (65%) reported discontinuation of PPI. Total acid exposure time (5.84 ± 4.63% to 2.83 ± 3.41%, P = 0.024) and number of reflux episodes (73.05 ± 19.34 to 37.55 ± 22.71, P < 0.001) decreased significantly after ARMA. Improved esophagogastric junction (EGJ) barrier function, including increased lower esophageal sphincter resting pressure (13.89 ± 10.78 mmHg to 21.68 ± 11.5 mmHg, P = 0.034), 4-second integrated relaxation pressure (5.75 ± 6.42 mmHg to 9.99 ± 5.89 mmHg, P = 0.020), and EGJ-contractile integral(16.42 ± 16.93 mmHg · cm to 31.95 ± 21.25 mmHg · cm, P = 0.016), were observed. Esophageal body contractility also increased significantly (distal contractile integral, 966.85 ± 845.84 mmHg · s · cm to 1198.8 ± 811.74 mmHg · s · cm, P = 0.023). Patients with symptom improvement had better pre-AMRA esophageal body contractility. Conclusions ARMA effectively improves symptoms and reflux burden, EGJ barrier function, and esophageal body contractility in patients with PPIdependent GERD during short-term evaluation. Longer follow-up to clarify the sustainability of ARMA is needed.

Background/Aims: Anti-reflux mucosal ablation (ARMA) is a promising endoscopic intervention for proton pump inhibitor (PPI)-dependent gastroesophageal reflux disease (GERD). However, the effect of ARMA on esophageal motility remains unclear. Methods: Twenty patients with PPI-dependent GERD receiving ARMA were prospectively enrolled. Comprehensive self-report symptom questionnaires, endoscopy, 24-hour impedance-pH monitoring, and high-resolution impedance manometry were performed and analyzed before and 3 months after ARMA. Results: All ARMA procedures were performed successfully. Symptom scores, including GerdQ (11.16 ± 2.67 to 9.11 ± 2.64, P = 0.026) and reflux symptom index (11.63 ± 5.62 to 6.11 ± 3.86, P = 0.001), improved significantly, while 13 patients (65%) reported discontinuation of PPI. Total acid exposure time (5.84 ± 4.63% to 2.83 ± 3.41%, P = 0.024) and number of reflux episodes (73.05 ± 19.34 to 37.55 ± 22.71, P < 0.001) decreased significantly after ARMA. Improved esophagogastric junction (EGJ) barrier function, including increased lower esophageal sphincter resting pressure (13.89 ± 10.78 mmHg to 21.68 ± 11.5 mmHg, P = 0.034), 4-second integrated relaxation pressure (5.75 ± 6.42 mmHg to 9.99 ± 5.89 mmHg, P = 0.020), and EGJ-contractile integral(16.42 ± 16.93 mmHg · cm to 31.95 ± 21.25 mmHg · cm, P = 0.016), were observed. Esophageal body contractility also increased significantly (distal contractile integral, 966.85 ± 845.84 mmHg · s · cm to 1198.8 ± 811.74 mmHg · s · cm, P = 0.023). Patients with symptom improvement had better pre-AMRA esophageal body contractility. Conclusions ARMA effectively improves symptoms and reflux burden, EGJ barrier function, and esophageal body contractility in patients with PPIdependent GERD during short-term evaluation. Longer follow-up to clarify the sustainability of ARMA is needed.

Background/Aims: Anti-reflux mucosal ablation (ARMA) is a promising endoscopic intervention for proton pump inhibitor (PPI)-dependent gastroesophageal reflux disease (GERD). However, the effect of ARMA on esophageal motility remains unclear. Methods: Twenty patients with PPI-dependent GERD receiving ARMA were prospectively enrolled. Comprehensive self-report symptom questionnaires, endoscopy, 24-hour impedance-pH monitoring, and high-resolution impedance manometry were performed and analyzed before and 3 months after ARMA. Results: All ARMA procedures were performed successfully. Symptom scores, including GerdQ (11.16 ± 2.67 to 9.11 ± 2.64, P = 0.026) and reflux symptom index (11.63 ± 5.62 to 6.11 ± 3.86, P = 0.001), improved significantly, while 13 patients (65%) reported discontinuation of PPI. Total acid exposure time (5.84 ± 4.63% to 2.83 ± 3.41%, P = 0.024) and number of reflux episodes (73.05 ± 19.34 to 37.55 ± 22.71, P < 0.001) decreased significantly after ARMA. Improved esophagogastric junction (EGJ) barrier function, including increased lower esophageal sphincter resting pressure (13.89 ± 10.78 mmHg to 21.68 ± 11.5 mmHg, P = 0.034), 4-second integrated relaxation pressure (5.75 ± 6.42 mmHg to 9.99 ± 5.89 mmHg, P = 0.020), and EGJ-contractile integral(16.42 ± 16.93 mmHg · cm to 31.95 ± 21.25 mmHg · cm, P = 0.016), were observed. Esophageal body contractility also increased significantly (distal contractile integral, 966.85 ± 845.84 mmHg · s · cm to 1198.8 ± 811.74 mmHg · s · cm, P = 0.023). Patients with symptom improvement had better pre-AMRA esophageal body contractility. Conclusions ARMA effectively improves symptoms and reflux burden, EGJ barrier function, and esophageal body contractility in patients with PPIdependent GERD during short-term evaluation. Longer follow-up to clarify the sustainability of ARMA is needed.

Connexin (Cx), a multigene-encoded transmembrane protein family, forms either gap junctions ( GJ) or hemichannels (HC) to mediate intercellular communication in plasma mem¬brane between adjacent cells or interacts with proteins by its car- boxyl terminal in the cytoplasm to participate in the process of tumor cell proliferation, apoptosis, necrosis, invasion, metasta¬sis, drug resistance and stem cell characteristics.However, mi- slocalization of Cx in cytoplasm or nucleus often occurs in many tumors, and involved in the occurrence and development of tumors.Subcellular localization of Cx is affected by post-transla- tional modifications, including phosphorylation, ubiquitination, and acetylation.In this paper the classification and function of Cx, the relationship between subcellular localization of Cx and tumorigenesis and the regulation of post-translational modifica¬tion on Cx are reviewed in order to provide new ideas for the study of Cx as a potential target for cancer therapy.

Aim To explore the apoptosis of small eell lung eancer ( SCLC ) eells HI688 and H446 induced by nitidine chloride and its possible mechanism.Methods The effect of nitidine chloride or cisplatin ( DDP ) on the activity of SCLC cells was detected by j J MTT method; the morphological changes of cells trea¬ted with nitidine chloride or DDP were observed by in- verted fluorescence microscope and HE staining; the effect of nitidine chloride or DDP on apoptosis was de¬tected by flow cytometry; the effect of apoptosis inhibi¬tor Z-VAD-FMK on apoptosis induced by nitidine chlo¬ride or DDP was detected by MTT method.The expres¬sions of Bax , Bcl-2, caspase-3 , PARP, p-PI3K and p- Akt in the cells treated with nitidine chloride or DDP were detected by Western blot.Results MTT results showed that the viability of SCLC cells was significantly reduced after 48 hours of treatment with nitidine chlo¬ ride; compared with DDP, nitidine chloride could in¬hibit SCLC cells with less IC50; inverted fluorescence microscope and HE staining showed that nitidine chlo¬ride could induce apoptosis in SCLC cells, similar to DDP; flow cytometry showed that nitidine chloride J J could induce apoptosis in SCLC cells.The results of MTT assay showed that the inhibitory effect of nitidine chloride on apoptosis of SCLC cells could be partially antagonized by apoptosis inhibitor Z-VAD-FMK.West¬ern blot results showed that, similar to DDP, nitidine chloride could inhibit the expression of PI3K and Akt, increase Bax, inhibit Be 1-2, and promote the cleavage of caspase-3 and PAH P.Conclusion Nitidine chlo¬ride can induce apoptosis of SCLC cells by inhibiting the activation of P13K and Akt.