To investigate the effect of GFP fused to C terminal of integrin alpha(IIb) on the biosynthesis and expression of alpha(IIb) beta(3) compound, the alpha(IIb) GFP expression plamid, named palpha(IIb) GFP, the cDNA of alpha(IIb) was constructed from p3.1-2b and fused to pEGFP-N1 in frame. When the sequence of palpha(IIb) GFP was confirmed by sequencing it was transferred to Chinese Hamster Ovary (CHO) cells with or without p3.1-3a expressing integrin beta(3). Then the expression of alpha(IIb) GFP fusion protein was confirmed by Western blot and then its subcellular localization was determined with laser confocal scanning microscopy. The results showed that the target gene was cloned into recombinant vector by restriction analysis and sequencing. Overexpression of the fusion protein in the transfected CHO cells was identified with Western blot. Subcellular localization analysis confirmed that alpha(IIb) GFP was expressed in CHO cells and could be transferred from endoplasmic reticulum to Golgi apparatus. It is concluded that the eukaryotic expression plasmid containing alpha(IIb) GFP fusion gene is successfully constructed. GFP fused to the cytoplasmic tail of integrin alpha(IIb) allows the normal expression of alpha(IIb) beta(3) in CHO cells.
Animals ; Blotting, Western ; CHO Cells ; Cricetinae ; Cricetulus ; Endoplasmic Reticulum ; metabolism ; Golgi Apparatus ; metabolism ; Green Fluorescent Proteins ; genetics ; metabolism ; Microscopy, Confocal ; Platelet Glycoprotein GPIIb-IIIa Complex ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism ; Transfection

To carry out the secretive expression of human 67 kD laminin receptor (67LR), recombinant expression plasmid pPIC9K-67LR was constructed by inserting of 67LR cDNA into yeast expression vector pPIC9K. The 67LR protein was expressed in Pichia pastoris after induced by methanol, and about 12.56 mg electrophoresis purity 67LR could be obtained after the purification of 1L culture using affinity chromatograph column. In vitro competitive binding assay showed that target protein has an excellent biological activity. The successful expression of 67LR has placed a solid foundation for the research on structure and functions of 67LR.
Genetic Vectors ; Humans ; Pichia ; genetics ; metabolism ; Plasmids ; genetics ; Receptors, Laminin ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; metabolism

Objective To evaluate the clinical effect of embolization of cerebral dural atreriovenous fistulas (cDAVF) of the eavemous sinus through the superior ophthalmic vein approach. Methods Twnety-seven patients with eDAVF of the cavernous sinus were embolized through the superior ophthalmic vein approach. Cerebral angiography and follow-up examination of the patients were performed to evaluate the effect ofernbolization. Results The fistulae showed complete angiographic disappearance in 15 patients, and 12 patients had blood velocity flow reduction at the fistula orifice. Ocular proptosis and chemosis deteriorated transiently in 11 patients after the procedure. The patients were followed-op for 3 to 48 months, and clinical cure was achieved in 17 patients, and 10 showed significant symptom relief. Conclusion cDAVF of the cavernous sinus can be effectively embolized through the superior ophthalmic vein approach.

Objective To analyze the A TP7A gene mutations in 2 related Chinese patients with Menkes disease (MD) and other members of the family and their hereditary features. Methods Two patients were clinically diagnosed as having MD. All 23 exons and exon-intron boundaries of ATP7A gene were polymerase chain reaction (PCR)-amplified and directly sequenced for genomic DNA extracted from the peripheral blood of both 2 patients and other members of the family; healthy controls were employed too. The mutations were proved by fluorescence quantitative PCR. Results Gross deletions from exon 2-12 were found in these 2 patients,respectively; their mothers,grandmother and aunt with normal phenotype carried those heterozygous mutations in the same site of A TP7A gene.Conclusions The 2 patients with MD are identified by gene and gross deletions from exon 2-12 are reported.

OBJECTIVETo study the effect of glycoprotein (GP) alpha II bA2334C mutation on the biosynthesis and expression of alpha II bbeta3 complex.

METHODSThe GP alpha II bA2334C eukaryotic expression plasmid pc3.1-2334M2b was constructed. Chinese hamster ovary (CHO) cells were transfected with the plasmid with or without integrin beta3 expression plasmid pc3.1-3a. The whole expression of alpha II bA2334C was confirmed by Western blot and the membrane expression was analyzed by flow cytometry. A newly constructed alpha II bA2334C GFP fusion protein expressing plasmid was used to determine its subcellular localization by laser confocal scanning microscopy.

RESULTSExpression of the mutant protein, alpha II bA2334C, in the transfected CHO cells was confirmed by Western blot with a lower rate of the mature type than the wild type control. The expression on membrane was only 25% of the normal. Subcellular localization analysis showed that alpha II bA2334C GFP was able to be expressed in CHO cells and could be transported from endoplasmic reticulum to Golgi apparatus.

CONCLUSIONSThe mutant alpha II bA2334C can be synthesized in CHO cells and form alpha II bbeta3 complex. However, only a small fraction of the premature alpha II bA2334C can be transported to Golgi apparatus and transformed to mature alpha II b. The possible pathogenesis of this type II thrombasthenia may be that the misfolded alpha II bA2334C is partially degraded in the endoplasmic reticulum causing lower expression of alpha II bbeta3 complex on the membrane and resulting in impared function of platelets than normal alpha II b.

Animals ; Biological Transport ; Blotting, Western ; CHO Cells ; Cricetinae ; Cricetulus ; Female ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Liposomes ; Microscopy, Confocal ; Middle Aged ; Mutation ; Plasmids ; genetics ; Platelet Glycoprotein GPIIb-IIIa Complex ; genetics ; metabolism ; Transfection

OBJECTIVETo compare the clinical outcomes of laparoscopic cryoablation (LCA) and laparoscopic partial nephrectomy (LPN) in the treatment of renal cell carcinoma (RCC).

METHODSBetween April 2005 and March 2009, 47 patients were treated with minimally invasive nephron sparing surgery (LPN or LCA) for RCC. The LCA group included 18 selected primary RCC cases (14 men and 4 women, mean age 63 years). There were 6 tumors located in the left, 11 located in the right and 1 located bilaterally. The maximum diameter of tumors was 1.5 - 5.0 cm (mean: 2.9 cm). The LPN group included 29 renal tumors patients (19 men and 10 women, mean age 61 years). The maximum diameter of tumors in this group was 2.0 - 4.5 cm (mean: 2.8 cm). Changes of hemoglobin (Hb), erythrocyte sedimentation rate (ESR), serum creatinine (SCr) and glomerular filtration rate (GFR) after operations were compared between LCA group and LPN group. The operative time, average intra-operative bleeding volume, postoperative hospital stay and incidence of postoperative complications of the 2 groups were analyzed and compared.

RESULTSThe 2 surgical procedures were both successful. There was no significant change of Hb, ESR, SCr and GFR after operations in LCA group and LPN group (P > 0.05). The operative time was (94 ± 29) min and (146 ± 45) min in LCA group and LPN group, respectively. The average estimated blood loss was (37 ± 20) ml and (274 ± 69) ml. The postoperative hospital stay was (4 ± 2) d and (10 ± 2) d. These differences between the 2 groups were significant (P < 0.01). No laparoscopic operative complications were noted in LCA group. Follow-up magnetic resonance imaging (MRI) at 1, 3, and 6 months identified the punched-out, nonenhancing, spontaneously resorbing, renal cryolesion. LCA group had completed a minimum follow-up of 6 months (mean 16, range 6 to 21 months). No evidence of local or port-site recurrence was found, and no patient developed metastatic disease. 3 - 36 months' (mean 20 months) follow-up showed no recurrence of tumors or metastatic disease in LPN group.

CONCLUSIONSLCA for RCC is an accurate and effective intervention with a relatively low incidence of complications, and is superior to LPN in operative time, intraoperative bleeding volume and postoperative recovery.

Adult ; Aged ; Carcinoma, Renal Cell ; surgery ; Cryosurgery ; methods ; Female ; Follow-Up Studies ; Humans ; Kidney Neoplasms ; surgery ; Laparoscopy ; Male ; Middle Aged ; Nephrectomy ; methods ; Retrospective Studies ; Treatment Outcome

OBJECTIVETo find the best treatment of penile strangulation and to analyze the sexual psychological factors of the patients.

METHODSWe retrospectively analyzed the experiences in removing foreign objects around the penis in 21 patients aged 19 - 61 years with the strangulation time varying from 10 hours to 4 days. The objects were mostly made of metal or plastics.

RESULTSAll the objects were successfully removed, 5 of them with the help of lubricant, 4 by aspirating the corpus cavernosum, 8 by shipping with pliers, 2 with the diamond-tipped dental drill, and the other 2, which virtually defied cutting, by aspirating the corpus cavernosum following degloving surgery.

CONCLUSIONIn removing foreign objects around the penis, simpler methods should be given precedence over more complex ones, and for those that virtually defy cutting, the best option is degloving surgery with particular attention to the survival of the penile skin flap.

Adult ; Foreign Bodies ; psychology ; surgery ; Humans ; Male ; Middle Aged ; Penis ; pathology ; surgery ; Retrospective Studies ; Young Adult

Objective: To explore the effect of Feng-liao-chang-wei-kang (FLCWK) on acute and chronic gastroenteritis, synergistic effect on the growth inhibitory effect with 5-fluorouracil (5-FU) on colorectal cancer and its underlying mechanisms. Methods: In the in vitro study, HT-29 cells were divided into 5-FU alone, FLCWK alone and coadministration groups. The MTT assay was used to analyze the proliferation of HT-29 cells at 24 h. Flow cytometry was used to observe the apoptosis, cycle of colorectal cancer HT-29 cells at 24 h. In the in vivo experiment, The subcutaneous transplantation tumor model of colorectal cancer HT-29-Luc was established with nude mice. All mice were randomly divided into 5-FU alone, FLCWK alone and coadministration groups according to body weight. During administration, the Interactive Video Information System small animal live imaging system was used to monitor the growth of subcutaneous transplantation in nude mice. The model of colitis-associated colorectal cancer (CACC) was established with BALB/c mice. BALB/c mice were randomly divided into the normal control group, the model control group, and the FLCWK group. At the end of the administration, the pathological status was detected by HE staining. Cell apoptosis of tumor tissue tumor and colon tissues were observed by TUNEL staining and TUNEL green fluorescence. The protein expression of Caspase 3, p-STAT3, Bcl-2, Bax and P-gp in tumor tissues tumor and colon tissues were tested by using immunohistochemical assay. Results: FLCWK and 5-FU coadministration suppressed HT-29 cell viability and induced S phase arrest and apoptosis compared to treatment with 5-FU alone. Furthermore, compared to treatment with 5-FU alone, coadministration of FLCWK and 5-FU obviously reduced tumor volume and weight and induced apoptosis through decreasing p-STAT3 and P-gp and increasing Caspase 3 protein expression in a murine xenograft tumor model. Moreover, the result revealed decreased number and size of tumors following FLCWK protective administration, downregulated p-STAT3 and Bcl-2 levels and upregulated Bax and Caspase 3 expression in mice with CACC. Conclusions: FLCWK has synergistic effects with 5-FU on colorectal cancer by suppressing the STAT3 pathway and downregulating P-gp expression. Furthermore, FLCWK administration suppresses CACC tumorigenesis by inhibiting the STAT3 pathway.