The vitamin K epoxide reductase complex subunit 1 (VKORC1), the rate-limiting enzyme for vitamin K recycling, is significantly down-regulated in the kidneys of urolithiasis patients. This study searched for direct evidence to define the inhibitory activity of VKORC1 against calcium oxalate (CaOx) crystal formation. In the experiment of VKORC1 overexpression, HK-2 cells were transfected with the pFLAG-CMV-7.1-VKORC1 plasmid as a pFLAG-CMV-7.1-VKORC1 transfection group or the pFLAG-CMV-7.1 plasmid as a pFLAG-CMV-7.1 control group. In the experiment of VKORC1 knockdown, HK-2 cells were transfected with the PGPU6/GFP/Neo-VKORC1shRNA-2 as a PGPU6/GFP/Neo-VKORC1shRNA-2 transfection group or the PGPU6/GFP/Neo-shRNA-NC plasmid as a PGPU6/GFP/Neo-shRNA-NC control group. The expression of VKORC1 in HK-2 cells was detected by real-time quantitative PCR and Western blotting. The CaOx crystal formation was observed under the laser-scanning confocal microscope. It was found that the expression levels of VKORC1 mRNA and protein were significantly higher in the pFLAG-CMV-7.1-VKORC1 transfection group than in the pFLAG-CMV-7.1 control group (P<0.01). The number of CaOx crystals in HK-2 cells incubated in fluorescently labeled CaOx monohydrate (COM) crystal medium for 48 h was 14±4 per field (100×) in the pFLAG-CMV-7.1-VKORC1 transfection group and 26±5 per field (100×) in the pFLAG-CMV-7.1 control group respectively under the laser-scanning confocal microscope. The amount of CaOx crystal aggregation and formation in the pFLAG-CMV-7.1-VKORC1 transfection group was significantly reduced as compared with the pFLAG-CMV-7.1 control group (P<0.05). The expression levels of VKORC1 mRNA and protein were significantly lower in the PGPU6/GFP/Neo-VKORC1shRNA-2 transfection group than in the PGPU6/GFP/Neo-shRNA-NC control group (P<0.05). The number of CaOx crystals in HK-2 cells incubated in fluorescently labeled COM crystal medium was 65±11 per field (100×) in the PGPU6/GFP/Neo-VKORC1shRNA-2 transfection group and 24±6 per field (100×) in the PGPU6/GFP/Neo-shRNA-NC control group respectively under the laser-scanning confocal microscope. The amount of CaOx crystal aggregation and formation in the PGPU6/GFP/Neo-VKORC1shRNA-2 transfection group was significantly increased as compared with the PGPU6/GFP/Neo-shRNA-NC control group (P<0.05). These findings suggested that the VKORC1 protein could inhibit CaOx salt crystallization, adhesion and aggregation. This research would help us to understand the mechanisms involving the interaction between crystallization and epithelial cells and the formation of CaOx.

ObjectiveTo probe the indication,treatment algorithm,anesthesia method,safety and efficacy ofextracorporealshockwavelithotripsy(ESWL)incombinationwithendoscopicretrograde cholangiopancreatography (ERCP) for pancreatic duct stones.MethodsThe patients with chronic pancreatitis and large pancreatic duct stones ( ＞ 5 mm diameter) and receiving ESWL and ERCP between March and July 2011 in Changhai Hospital were prospective studied.The third generation of extracorporeal shockwave lithotripsy was applied in ESWL,and the localization of stone was determined by X-ray.No more than 5000 shocks were given per session,and ESWL was performed continuously till the calculi were fragmented,and then was cleared by ERCP.ResultsA total of 100 patients underwent ESWL during the 5 months,among whom 84 patients received ERCP treatment and 41 cases failed to deep cannulation (41/84,48.8％ ).Multiple stones were seen in 83 patients.Ninety five patients had radio-opaque stones,two patients had radiolucent calculi,while three patients had both radio-opaque and radiolucent stones.Seventy five percent,14％ and 11％ stones were located in pancreatic head,pancreatic head and body,pancreatic body and tail,respectively.A total of 175 ESWL procedures were performed,43 patients needed 2 or more sessions for successful fragmentation.Anesthesia method was mainly intravenous sedation,accounting for 96％ (168/175).ERCP was successful in 96 patients after ESWL,only 4 patients failed after ESWL. Forty one cases which failed ERCP procedures before ESWL underwent ERCP,and 37 patients (90.2％) achieved successful cannulation.Successful fragmentation ratewas 100％.Complete clearance was achieved in 78 patients,and complication rate of post-ERCP pancreatitis,fever was 1.71％ (n =3 ),0.57％ (n =1 ),and the overall complication rate was 2.28％.Conclusions ESWL is an effective,safe and necessary modality for fragmentation of large PD stones in the management of minimal invasive treatment of chronic pancreatitis.

The vitamin K epoxide reductase complex subunit 1 (VKORC1), the rate-limiting enzyme for vitamin K recycling, is significantly down-regulated in the kidneys of urolithiasis patients. This study searched for direct evidence to define the inhibitory activity of VKORC1 against calcium oxalate (CaOx) crystal formation. In the experiment of VKORC1 overexpression, HK-2 cells were transfected with the pFLAG-CMV-7.1-VKORC1 plasmid as a pFLAG-CMV-7.1-VKORC1 transfection group or the pFLAG-CMV-7.1 plasmid as a pFLAG-CMV-7.1 control group. In the experiment of VKORC1 knockdown, HK-2 cells were transfected with the PGPU6/GFP/Neo-VKORC1shRNA-2 as a PGPU6/GFP/Neo-VKORC1shRNA-2 transfection group or the PGPU6/GFP/Neo-shRNA-NC plasmid as a PGPU6/GFP/Neo-shRNA-NC control group. The expression of VKORC1 in HK-2 cells was detected by real-time quantitative PCR and Western blotting. The CaOx crystal formation was observed under the laser-scanning confocal microscope. It was found that the expression levels of VKORC1 mRNA and protein were significantly higher in the pFLAG-CMV-7.1-VKORC1 transfection group than in the pFLAG-CMV-7.1 control group (P<0.01). The number of CaOx crystals in HK-2 cells incubated in fluorescently labeled CaOx monohydrate (COM) crystal medium for 48 h was 14±4 per field (100×) in the pFLAG-CMV-7.1-VKORC1 transfection group and 26±5 per field (100×) in the pFLAG-CMV-7.1 control group respectively under the laser-scanning confocal microscope. The amount of CaOx crystal aggregation and formation in the pFLAG-CMV-7.1-VKORC1 transfection group was significantly reduced as compared with the pFLAG-CMV-7.1 control group (P<0.05). The expression levels of VKORC1 mRNA and protein were significantly lower in the PGPU6/GFP/Neo-VKORC1shRNA-2 transfection group than in the PGPU6/GFP/Neo-shRNA-NC control group (P<0.05). The number of CaOx crystals in HK-2 cells incubated in fluorescently labeled COM crystal medium was 65±11 per field (100×) in the PGPU6/GFP/Neo-VKORC1shRNA-2 transfection group and 24±6 per field (100×) in the PGPU6/GFP/Neo-shRNA-NC control group respectively under the laser-scanning confocal microscope. The amount of CaOx crystal aggregation and formation in the PGPU6/GFP/Neo-VKORC1shRNA-2 transfection group was significantly increased as compared with the PGPU6/GFP/Neo-shRNA-NC control group (P<0.05). These findings suggested that the VKORC1 protein could inhibit CaOx salt crystallization, adhesion and aggregation. This research would help us to understand the mechanisms involving the interaction between crystallization and epithelial cells and the formation of CaOx.
Apoptosis ; drug effects ; Blotting, Western ; Calcium Oxalate ; chemistry ; metabolism ; pharmacology ; Cell Line ; Crystallization ; Dose-Response Relationship, Drug ; Flow Cytometry ; Gene Expression ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Microscopy, Confocal ; RNA Interference ; Reverse Transcriptase Polymerase Chain Reaction ; Time Factors ; Transfection ; Vitamin K Epoxide Reductases ; genetics ; metabolism

OBJECTIVETo observe the effect of thermotherapy on the intracellular adriamycin concentration and apoptosis of Raji cells in vitro.

METHODSThe working concentration of adriamycin against Raji cells was determined with MTT assay. Raji cells were subjected to thermotherapy (at 40 degrees Celsius;, 41 degrees Celsius; or 42 degrees Celsius;) and chemotherapy with adriamycin alone or in conjunction, and the cell survival rates were determined 48 h after the treatment. The cell growth inhibition effect of the treatment was evaluated with MTT assay, and the apoptotic rates of Raji cells and alteration of intracellular adriamycin concentration were determined by flow cytometry.

RESULTSThe IC(50) of adriamycin was defined as the working concentration in the experiment. Thermotherapy at 40, 41 and 42 degrees Celsius; for 60 min in conjuction with chemotherapy significantly inhibited the growth of Raji cells (P<0.01). The results of flow cytometry showed that thermotherapy and adriamycin chemotherapy, used either alone or in combination, significantly increased the apoptotic rates of Raji cells (P<0.01), and thermotherapy remarkably increased the intracellular concentration of adriamycin.

CONCLUSIONAdriamycin chemotherapy combined thermotherapy for 60 min can increase the intracellular concentration of adriamycin and the apoptosis rates of Raji cells.

Antibiotics, Antineoplastic ; pharmacology ; Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Combined Modality Therapy ; Doxorubicin ; metabolism ; pharmacology ; Flow Cytometry ; Hot Temperature ; Humans ; Hyperthermia, Induced ; Inhibitory Concentration 50 ; Intracellular Space ; metabolism ; Lymphoma ; drug therapy ; metabolism ; pathology

LG1 strain of avian influenza virus H9N2 was passaged continuously for 40 generations in chicken embryos with anti-LG1 maternal antibodies in 4 parallel experiments, of which 3 experiments had a stable mutation of "G" to "A" at #99 of the neuraminidase gene(NA)from the 20th passage resulting in a change of Met to Ile and 2 had a stable mutation of "A" to "G" at #473 of the NA gene from the 30th passage resulting in a change of Asn to Ser which occurred in the 50th passage of another experiment. Eighty continuous passages in chicken embryos without antibody did not have the same mutation, indicating that the mutations of the 2 positions were associated with selective pressure of antibodies. Analysis of the ratios of nonsynonium (NS) vs synonium (S) mutations of nucleic acids demonstrated that NS/S of 4 parallel experiments with antibodies was 4.6 (32/7) compared with 2.0 (16/8) of the 2 experiments without antibodies and this significant difference implied the selective pressure of antibodies.
Animals ; Antibodies, Viral ; immunology ; Chick Embryo ; Influenza A Virus, H9N2 Subtype ; genetics ; immunology ; Mutation ; Neuraminidase ; genetics

The purpose of this study was to compare the whole genome sequences and replication dynamics in cell cultures of two Avian leukosis viruses of subgroup B (ALV) isolates, SDAU09E3 and SDAU09C2. Comparison of the amino acid sequences indicated that the gp85 identity of these two subgroup B isolates was 95.4%, the identity with other three ALV-B reference strains was 91.0%-94.9%, and less than 87.9% with ALV subgroup A, C, D, E and J. Comparison of the nucleotide sequence of gag and pol genes indicated that homologies of gag gene and pol gene of these two ALV-B isolates with all compared reference strains of different subgroups were above 93%. Homologies of LTR sequence of these two ALV-B isolates with other exogenous ALVs subgroups A, B, C, D and J were 72.6%-88.3%, but only 51.5% when compared with endogenous ALV subgroup E. The identity of LTR between these two ALV-B strains was only 74.8%, which was far lower than the identity of other genes. The identity of U3 region of LTR between these two ALV-B isolates was only 68.8% and there were obvious differences in the number CAAT Boxes. Replication dynamics in DF-1 cell indicated that the value of TCID50 was similar between 2 isolates but the concentration of nucleocapsid protein p27 antigen of SDAU09E3 was significantly higher than SDAU09C2 in cell culture supernatant, which indicated there was no parallel relationship between p27 antigen concentration and infectious virus particles. Whether such difference was resulted from the diversity of U3 region of LTR, further studies with their recombinant infectious clones is necessary.
Animals ; Antibodies, Viral ; immunology ; Avian Leukosis Virus ; classification ; genetics ; physiology ; Base Sequence ; Cell Line ; Cells, Cultured ; Chick Embryo ; Chickens ; Genome, Viral ; genetics ; Molecular Sequence Data ; Phylogeny ; Poultry Diseases ; virology ; Sequence Alignment ; Sequence Homology, Nucleic Acid ; Viral Matrix Proteins ; genetics ; Virus Replication ; physiology