OBJECTIVETo develop a targeting protein for Xenopus kinesin-like protein 2 (TPX2) C' terminal SBP-3 x Flag-tagged HCT 116 cell model.

METHODSHomologous arms were amplified by polymerase chain reaction (PCR), and then the adeno-associated virus (AAV) -targeting vector of TPX2 was constructed. HCT 116 cells were targeted after the viruses were packaged. Positive cell clones with neomycin resistance gene were obtained by G418 and PCR screening. Finally, the neomycin gene cassette was excised after the targeted clones were infected with adenovirus expressing Cre-recombinase, and the TPX2 C' terminal SBP and 3 x Flag endogenous double-tagged HCT 116 cells were obtained by PCR screening.

RESULTSTwo positive cell clones with neomycin resistance gene were obtained by PCR screening. The positive clones with neomycin resistance gene excised were obtained by Cre adenovirus infection, and the knock-in of SBP-3 x Flag gene was verified by Western blot analysis.

CONCLUSIONThe TPX2 C' terminal SBP-3 x Flag tagged HCT 116 cell model was successfully established.

Cell Cycle Proteins ; genetics ; Colorectal Neoplasms ; genetics ; pathology ; Dependovirus ; genetics ; Gene Targeting ; Genetic Vectors ; HCT116 Cells ; Humans ; Microtubule-Associated Proteins ; genetics ; Nuclear Proteins ; genetics

OBJECTIVETo develop a specific SARS virus-targeted antibody preparation for emergent prophylaxis and treatment of SARS virus infection.

METHODSBy using phage display technology, we constructed a naive antibody library from convalescent SARS patient lymphocytes. To obtain the neutralizing antibody to SARS virus surface proteins, the library panning procedure was performed on purified SARS virions and the specific Fab antibody clones were enriched by four rounds of repeated panning procedure and screened by highthroughput selection. The selected Fab antibodies expressed in the periplasma of E. coli were soluble and further purified and tested for their binding properties and antiviral function to SARS virus. The functional Fab antibodies were converted to full human IgG antibodies with recombinant baculovirus/insect cell systems and their neutralizing activities were further determined.

RESULTSAfter four rounds of the panning, a number of SARS-CoV virus-targeted human recombinant Fab antibodies were isolated from the SARS patient antibody library. Most of these were identified to recognize both natural and recombinant SARS spike (S) proteins, two Fab antibodies were specific for the virus membrane (M) protein, only one bound to SARS-CoV nucleocapsid protein. The SARS-CoV S and M protein-targeted Fab or IgG antibodies showed significant neutralizing activities in cytopathic effect (CPE) inhibition neutralization test, these antibodies were able to completely neutralize the SARS virus and protect the Vero cells from CPE after virus infection. However, the N protein-targeted Fab or IgG antibodies failed to neutralize the virus. In addition, the SARS N protein-targeted human Fab antibody reacted with the denatured N proteins, whereas none of the S and M protein specific neutralizing antibodies did. These results suggested that the S and M protein-specific neutralizing antibodies could recognize conformational epitopes which might be involved in the binding of virions to cellular receptors and the fusion activity of the virus.

CONCLUSIONThe SARS-CoV spike protein and membrane proteins are able to elicite efficient neutralizing antibodies in SARS patients. The neutralizing antibodies we generated in this study may be more promising candidates for prophylaxis and treatment of SARS infection.

Amino Acid Sequence ; Animals ; Antibodies, Viral ; immunology ; Cercopithecus aethiops ; Humans ; Membrane Glycoproteins ; immunology ; Neutralization Tests ; Peptide Library ; Protein Binding ; Protein Engineering ; Recombinant Proteins ; immunology ; SARS Virus ; immunology ; Severe Acute Respiratory Syndrome ; immunology ; virology ; Spike Glycoprotein, Coronavirus ; Vero Cells ; Viral Envelope Proteins ; immunology ; Viral Matrix Proteins ; immunology

BACKGROUNDAlthough thrombolytic therapy with rescue percutaneous coronary intervention (PCI) is a common treatment strategy for ST-segment elevation acute myocardial infarction (STEMI), scant data are available on its efficacy relative to primary PCI, and comparison was therefore the aim of this study.

METHODSThis multicenter, open-label, randomized, parallel trial was conducted in 12 hospitals on patients (age < or = 70 years) with STEMI who presented within 12 hours of symptom onset (mean interval > 3 hours). Patients were randomized to three groups: primary PCI group (n = 101); recombinant staphylokinase (r-Sak) group (n = 104); and recombinant tissue-type plasminogen activator (rt-PA) group (n = 106). For all patients allocated to the thrombolytic therapy arm, coronary angiography was performed at 90 minutes after drug therapy to confirm infarct-related artery (IRA) patency; rescue PCI was performed in cases with TIMI flow grade < or = 2. Bare-metal stent implantation was planned for all patients.

RESULTSAfter randomization it required an average of 113.4 minutes to start thrombolytic therapy (door-to-needle time) and 141.2 minutes to perform first balloon inflation in the IRA (door to balloon time). Rates of IRA patency (TIMI flow grade 2 or 3) and TIMI flow grade 3 were significantly lower in the thrombolysis group at 90 minutes after drug therapy than in the primary PCI group at the end of the procedure (70.5% vs. 98.0%, P < 0.0001, and 53.0% vs. 85.9%, P < 0.0001, respectively). Rescue PCI with stenting was performed in 117 patients (55.7%) in the thrombolytic therapy arm. Rates of patency and TIMI flow grade 3 were still significantly lower in the rescue PCI than in the primary PCI group (88.9% vs. 97.9%, P = 0.0222, and 68.4% vs. 85.0%, P = 0.0190, respectively). At 30 days post-therapy, mortality rate was significantly higher in the thrombolysis combined with rescue PCI group than in primary PCI group (7.1% vs. 0, P = 0.0034). Rates of death/MI and bleeding complications were significantly higher in the thrombolysis with rescue PCI group than in the primary PCI group (10.0% vs. 1.0%, P = 0.0380, and 28.10% vs. 8.91%, P = 0.0001, respectively).

CONCLUSIONSThrombolytic therapy with rescue PCI was associated with significantly lower rates of coronary patency and TIMI flow grade 3, but with significantly higher rates of mortality, death/MI and hemorrhagic complications at 30 days, as compared with primary PCI in this group of Chinese STEMI patients with late presentation and delayed treatments.

Aged ; Angioplasty, Balloon, Coronary ; Coronary Angiography ; Female ; Fibrinolytic Agents ; therapeutic use ; Humans ; Male ; Middle Aged ; Myocardial Infarction ; drug therapy ; therapy ; Thrombolytic Therapy