Adolescent ; Adult ; Cross Infection ; microbiology ; Female ; Humans ; Male ; Microbial Sensitivity Tests ; Middle Aged ; Organophosphate Poisoning ; Pesticides ; poisoning ; Pneumonia ; microbiology ; Respiration, Artificial ; Respiratory Insufficiency ; chemically induced ; therapy

This study is to construct a chimeric human/porcine BDD-FVIII (BDD-hpFVIII) containing the substituted porcine A1 and A3 domains which proved to have a pro-secretory function. By exploring Ssp DnaB intein's protein trans-splicing a dual-vector was adopted to co-transfer the chimeric BDD-hpFVIII gene into cultured COS-7 cell to observe the intracellular BDD-hpFVIII splicing by Western blotting and secretion of spliced chimeric BDD-hp FVIII protein and bio-activity using ELISA and Coatest assay, respectively. The dada showed that an obvious protein band of spliced BDD-hpFVIII can be seen, and the amount of spliced BDD-hpFVIII protein and bio-activity in the supernatant were up to (340 +/- 64) ng x mL(-1) and (2.52 +/- 0.32) u x mL(-1) secreted by co-transfected cells which were significantly higher than that of dual-vector-mediated human BDD-FVIII gene co-transfection cells [(93 +/- 22) ng x mL(-1), (0.72 +/- 0.13) u x mL(-1)]. Furthermore, a spliced BDD-hpFVIII protein and activity can be detected in supernatant from combined cells separately transfected with intein-fused BDD-hpFVIII heavy and light chain genes indicating that intein-mediated BDD-hpFVIII splicing occurs independently of cellular mechanism. It provided evidence for enhancing FVIII secretion in the research of animal models using intein-based dual vector for the delivery of the BDD-hpFVIII gene.
Animals ; COS Cells ; Cercopithecus aethiops ; Factor VIII ; genetics ; metabolism ; secretion ; Genetic Vectors ; Humans ; Inteins ; Peptide Fragments ; genetics ; metabolism ; secretion ; Plasmids ; Protein Splicing ; Swine ; Trans-Splicing ; Transfection

As synthesized by vascular endothelial cells and megakaryocytes, the von Willebrand factor (vWF) plays an important hemostatic role in the binding to and stabilizing blood coagulation factor VIII (FVIII) and preventing its enzymatic degradation. Our recent work demonstrated intein can efficiently ligate BDD-FVIII (B-domaim deleted FVIII) posttranslationally by protein trans-splicing after transfer of split BDD-FVIII gene by a dual-vector system. In this study we investigated the effect of vWF on secretion and activity of intein-ligated BDD-FVIII. We observed the levels of full-length BDD-FVIII antigen secreted into culture supernatant by ELISA and their activity by Coatest assay after transfection of cultured 293 cells with intein-fused BDD-FVIII heavy- and light-chain genes simultaneously with the vWF gene co-transfected. The data showed that the amount of full-length BDD-FVIII protein and their bioactivity in vWF gene co-transfected cell supernatant were 235 +/- 21 ng x mL(-1) and 1.98 +/- 0.2 u x mL(-1), respectively, greater than that of non-vWF co-transfected cell (110 +/- 18) ng x mL(-1) and 1.10 +/- 0.15 u x nL(-1)) or just BDD-FVIII gene transfected control cell (131 +/- 25 ng x mL(-1) and 1.22 +/- 0.18 u x mL(-1)) indicating the benefit of vWF gene co-transfection in the secretion and activity of intein-spliced BDD-FVIII protein. It provided evidence that vWF gene co-transfer may be useful to improve efficacy of gene therapy for hemophilia A in protein splicing-based split FVIII gene transfer.
Factor VIII ; genetics ; metabolism ; secretion ; Genetic Therapy ; Genetic Vectors ; HEK293 Cells ; Hemophilia A ; therapy ; Humans ; Inteins ; Peptide Fragments ; genetics ; metabolism ; secretion ; Plasmids ; Protein Splicing ; Trans-Splicing ; Transfection ; von Willebrand Factor ; genetics ; metabolism ; physiology

China ; Genotype ; Measles virus ; genetics ; isolation & purification ; Molecular Epidemiology ; methods ; Nucleoproteins ; genetics ; Polymerase Chain Reaction

In our recent study by exploring an intein-based dual-vector to deliver a B-domain-deleted FVIII (BDD-FVIII) gene, it showed that covalently ligated intact BDD-FVIII molecules with a specific coagulant activity could be produced from expressed heavy and light chains by protein trans-splicing. Here, we assessed the hypothesis that the efficiency of trans-splicing may be increased by adding to the intein sequences a pair of leucine zippers that are known to bring about specific and strong protein binding. The intein-fused heavy and light chain genes were co-transferred into cultured COS-7 cells using a dual-vector system. After transient expression, the intracellular BDD-FVIII splicing was observed and the spliced BDD-FVIII and bioactivity secreted to culture media were quantitatively analyzed. An enhanced splicing of BDD-FVIII with decreased protein precursors from gene co-transfected cells was observed by Western blotting. The amount of spliced BDD-FVIII and bioactivity secreted to the culture media were 106 +/- 12 ng x mL(-1) and 0.89 +/- 0.11 U x mL(-1) analyzed by ELISA and Coatest method respectively, which was greater than leucine zipper free intein-fused heavy and light chain genes co-transfected cells (72 +/- 10 ng x mL(-1) and 0.62 +/- 0.07 U x mL(-1)). The activity of cellular mechanism-independent protein splicing was also improved, as showed by the increasing of spliced BDD-FVIII and bioactivity in culture media from combined cells separately transfected with heavy and light chain genes which was 36 +/- 11 ng x mL(-1) and 0.28 +/- 0.09 U x mL(-1). It demonstrated that the leucine zippers could be used to increase the efficiency of protein trans-splicing to improve the efficacy of a dual-vector mediated BDD-FVIII gene delivery by strengthening the interaction between the two intein-pieces fused to heavy and light chains. It provided evidence for further study in animal model using a dual-adeno-associated virus vector to deliver FVIII gene in vivo.
Animals ; COS Cells ; Cercopithecus aethiops ; Factor VIII ; chemistry ; genetics ; metabolism ; Genetic Vectors ; Inteins ; Leucine Zippers ; Peptide Fragments ; chemistry ; genetics ; metabolism ; Protein Splicing ; Trans-Splicing ; Transfection

Although two chain transfering separately could be used to overcome the volume limitation of adeno-associated virus vectors (AAV) in coagulation factor VIII (FVIII) gene delivery, it leads to chain imbalance for inefficient heavy chain secretion. In this study we aimed to improve the efficacy of two chain strategy in FVIII gene delivery through the degradation of glucose-regulated protein 78 (GRP78) known as a protein chaperone in endoplasmic reticulum (ER) by deoxynivalenol (DON) to decrease GRP78-bound FVIII heavy chain. By treating the two-chain gene transduced 293 cells with DON, the heavy chain (HC) secretion and FVIII bioactivity were observed. Data showed that 293 cells after three hours post-treatment with DON at a concentration of 500 ng mL(-1) resulted in obvious decrease the level of GRP78 but no effect on the cell proliferation. The HC secreted from DON-treated cells transfected with HC gene alone was 59 +/- 11 ng mL(-1), higher than that secreted by control cells (15 +/- 4 ng mL(-1)), and the HC secretion was further increasing to 146 +/- 34 ng mL(-1) in light chain (LC) gene co-transfected cells with an activity measured up to 0.66 +/- 0.15 U mL(-1), also greater than control cells (76 +/- 17 ng mL(-1) and 0.35 +/- 0.09 U mL(-1)). Taken together, these data suggest that DON-mediated GRP78 down-regulation could improve the efficacy of two-chain FVIII gene transfering by facilitating HC secretion, providing an experimental basis for in vivo dual-AAV application in FVIII gene delivery.
Cell Proliferation ; Down-Regulation ; Factor VIII ; chemistry ; genetics ; secretion ; Gene Transfer Techniques ; HEK293 Cells ; Heat-Shock Proteins ; metabolism ; Humans ; Transfection ; Trichothecenes ; pharmacology

Abdomen ; Hernia ; Humans ; Prostheses and Implants ; Prosthesis Implantation ; Treatment Outcome

OBJECTIVETo investigate the effects of montelukast on atherosclerosis and monocyte chemoattractant protein-1 expression in a hypercholesterolemic rabbit model.

METHODSThirty four male New Zealand white rabbits were randomized into four groups including normal control group (n = 6), placebo group (n = 8), atorvastatin group (1.5 mgxkg(-1)xd(-1), beginning at 8(th) weeks for 4 weeks, n = 10) and montelukast group (1 mgxkg(-1)xd(-1), beginning at 8(th) weeks for 4 weeks, n = 10). Rabbits except those in normal control group were fed a high cholesterol diet for 12 weeks. Serum lipids were measured at 0, 8 and 12 weeks after intervention. The intima/media ratio, percentages of macrophages or smooth muscle cells in intima and the expression of MCP-1 mRNA were examined.

RESULTSAtherosclerosis was evidenced in placebo group and atorvastatin or montelukast treatment significantly reduced neointima (0.32 +/- 0.12 and 0.34 +/- 0.10 vs. 1.12 +/- 0.36, P < 0.05) and macrophage content [(9.8 +/- 4.6)% and (11.2 +/- 3.7)% vs. (34.6 +/- 8.8)%, P < 0.05], increased SMC content [(18.6 +/- 6.9)% and (19.2 +/- 8.6)% vs. (5.2 +/- 2.3)%, P < 0.05] and inhibited expression of MCP-1 mRNA (0.42 +/- 0.08 and 0.40 +/- 0.06 vs. 2.36 +/- 0.48, P < 0.01). Montelukast had similar anti-atherogenetic effects as atorvastatin but had no influence on plasma lipids.

CONCLUSIONSMontelukast could attenuate atherosclerosis in this hypercholesterolemic rabbit model which might be attributed to its anti-inflammatory effects.

Animals ; Atherosclerosis ; metabolism ; Chemokine CCL2 ; metabolism ; Hypercholesterolemia ; Macrophages ; metabolism ; Rabbits ; Tunica Intima

OBJECTIVETo evaluate the safety and long-term effect of recombinant human epithelial growth factor (rhEGF) on deep partial-thickness burn wounds.

METHODSThirty-seven burn patients were enrolled in this study and were observed by randomized, double-blinded and placebo-controlled protocol. An area of deep partial-thickness burn wounds from each patient was divided into control (C) and treatment (T) portions. The wound in C was treated with normal saline while that in T with rhEGF. The patients were followed-up for 1 and 4 years after wound healing. The healed wounds were evaluated by modified Vancouver scar scale in terms of scar index (SI).

RESULTS1 year after wound healing, it was found that the SI in T group (7.19 +/- 1.67) was obviously lower than that in C group (8.92 +/- 1.78, P < 0.01). The SI in T group (6.12 +/- 1.54) was still evidently lower than that in C group (8.09 +/- 1.81, P < 0.01) four years after wound healing. There were no signs of development of tumor or cancer in all the tested burn wound areas.

CONCLUSIONExternal application of rhEGF might be beneficial to the healing quality of deep partial-thickness burn wound with less scar formation and better long-term effects, and it is safe.

Adult ; Burns ; drug therapy ; Double-Blind Method ; Epidermal Growth Factor ; therapeutic use ; Female ; Follow-Up Studies ; Humans ; Male ; Recombinant Proteins ; therapeutic use ; Treatment Outcome ; Wound Healing ; drug effects ; Young Adult

OBJECTIVEElectron microscopical study of infected cells to identify the pathogenic agent of SARS.

METHODSVero E6 cells infected with lung autopsy samples or nasopharyngeal swabs from SARS patients of Beijing and Guangzhou were inoculated. The supernatant and cultured cells exhibiting identifiable cytopathic effect (CPE) were prepared for electron microscopic study.

RESULTSExamination of CPE cells on thin-section revealed characteristic coronavirus particles within the cisternae of endoplasmic reticulum, Golgi apparatus, vesicles and extracellular space. They were mainly spherical or oval in shape, annular or dense, about 80 nm in diameter. Negative-stain electron microscopy identified coronavirus particles in culture supernatant, 80 - 120 nm in diameter, with club-shaped surface projections. Elongated, rod-, kidney- or other irregular shaped virons with the size of 100 - 200 nm by 60 - 90 nm were also found in the cultured cells infected with the lung samples from the Guangdong patients. Infectious virons entered cells by endocytosis or membrane fusion and released through a budding process.

CONCLUSIONThese data indicate a novel coronavirus as the causative agent of SARS. Most viral particles showed typical characteristics of coronavirus. The potential role of special shape viruses is expected to be further investigated.

Animals ; Cercopithecus aethiops ; Humans ; Microscopy, Electron ; SARS Virus ; ultrastructure ; Severe Acute Respiratory Syndrome ; virology ; Vero Cells