According to the requirements of the regulatory authorities, degree of modification (DP) should be included in the characterisation of the PEGylated protein drug substance, and is one of the critical quality attributes for quality control. In this study, based on the fundamental assumption that the refractive index (RI) signal and the ultraviolet (UV) signal of PEGylated protein are equal to the sum of the corresponding signal produced by the polyethylene glycol (PEG) and protein parts of the conjugates in their uncoupled state, we developed a method to determine the DP of PEGylated recombinant human growth hormone (inpegsomatropin). In this method, 20 μL of 1 mg·mL^-1 human growth hormone (hGH) standard, 2 mg·mL^-1 PEG reference substance and 1 mg·mL^-1 drug substance solution were each injected to size exclusion chromatographic (SEC) column for separation, detected with ultraviolet and refractive index (UV-RI) detectors in series. Finally, the DP was calculated as the formula derived from the fundamental assumption. The developed SEC-UV-RI method showed good specificity, repeatability (RSD = 0.63%, n = 9) and accuracy, with a recovery of 100.0%, compared with the result obtained from the simultaneously established classical acid hydrolysis method, demonstrating pretty handleability and accessibility, and is appropriate for quality control test of DP for this drug substance.

Animals ; Aorta ; pathology ; Apolipoproteins E ; deficiency ; Atherosclerosis ; metabolism ; pathology ; Female ; Glycosides ; isolation & purification ; pharmacology ; Intercellular Adhesion Molecule-1 ; metabolism ; Mice ; Plants, Medicinal ; chemistry ; Polygonum ; chemistry ; Vascular Cell Adhesion Molecule-1 ; metabolism

OBJECTIVETo construct a eukaryotic expression vector for expressing hepatitis C virus (HCV) recombinant NS5B-EGFP fusion protein and obtain a stable transfected HepG2 cell line.

METHODSThe coding region of NS5B gene of HCV was amplified by PCR and was digested by Xho I/Kpn I. This fragment was inserted into pEGFPN3 with T4 ligase and transformed E. coli TG1. The positive recombinant plasmid was selected, then the recombinant plasmid was transfected into HepG2 cell by Lipofectin AMINE 2000. Cells containing stable transformants were selected by the ability of resistance to G418 and isolated with a limited dilution. The stable transfected cell line expressing high level NS5B-EGFP fusion protein was obtained.

RESULTSThe eukaryotic expression vector named pEGFPN3-ns5b was successfully constructed and the stable transfected HepG2 cell line expressing NS5B-EGFP fusion protein was obtained.

CONCLUSIONThe stable transfected HepG2 cell line could express NS5B-EGFP fusion protein, could be used for anti-HCV infection with ns5b gene as the target.

Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Microscopy, Fluorescence ; Recombinant Fusion Proteins ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection ; Viral Nonstructural Proteins ; genetics ; metabolism

OBJECTIVETo analyze the CT image features of pneumonic-type lung cancer and to reduce misdiagnosis.

METHODSThe CT findings of 46 patients with pneumonic-type lung cancer were retrospectively reviewed, and CT image in the differential diagnosis of this special kind of disease was evaluated.

RESULTSAccording to the extent of lesion, these cases were divided into two groups: multi-lobe consolidation group and single lobe consolidation group. The lesions in the latter group located in the upper, middle or lower lobe, respectively. Twenty-nine cases had homogeneous consolidation lesion, 14 cases showed single or multiple cysts and cavities in the lesions, 3 cases exhibited localized low density in the lesion. Forty-one cases shown the sign of air bronchogram with presentation of narrow air bronchogram in 25 of those. Forty cases showed well or ill defined ground-glass opacitiy surrounding the consolidation lesion. Fifteen cases had multi-nodules or opacities distributed in centrilobular or centric bronchiolar location. Of the 30 patients who received contrast medium, 23 showed distinct enhancement, and 7 showed indistinct enhancement with a positive CT angiogram.

CONCLUSIONCT findings including lower lobe distribution, homogeneous consolidation, narrow air bronchogram, well defined ground-glass and CT angiogram are helpful in differentiating pneumonic-type lung cancer from various kinds of infection. However, most of CT manifestations of pneumonic-type lung cancer are not specific. Therefore, it's necessary to combine CT findings with other clinical data when making diagnosis.

Adenocarcinoma ; diagnostic imaging ; Adenocarcinoma, Bronchiolo-Alveolar ; diagnostic imaging ; Adenocarcinoma, Mucinous ; diagnostic imaging ; Adenocarcinoma, Papillary ; diagnostic imaging ; Adult ; Aged ; Diagnosis, Differential ; Diagnostic Errors ; Female ; Follow-Up Studies ; Humans ; Lung ; diagnostic imaging ; Lung Neoplasms ; diagnostic imaging ; Male ; Middle Aged ; Pneumonia, Bacterial ; diagnostic imaging ; Radiographic Image Enhancement ; Tomography, X-Ray Computed ; methods ; Tuberculosis, Pulmonary ; diagnostic imaging

OBJECTIVETo identify and characterize the epitope associated with the virus attachment protein (VAP) of hantaan virus.

METHODSThe monoclonal antibody 3G1 was used as the ligand to biospan from a phage-displayed 12-amino acid peptide library, then the positive phage clones were chosen and sequenced. The amino acid sequences of them were compared with that of hantaan virus G2 in homology. The characteristics of positive phage were studied by IFA and ELISA. A decapeptide combining to cell membrane was observed under laser scanning confocal microscope (LSCM).

RESULTSThe conservative motif PX(1-2) HX(0-2) H displaying on positive clones shared homologous amino acid sequence with G2 96YPWHTAKCHY105.

CONCLUSIONG2 96YPWHTAKCHY105 might play some roles in virus binding to host cell, and might be a possible key epitope of hantaan virus VAP.

Animals ; CHO Cells ; Cell Membrane ; metabolism ; Cercopithecus aethiops ; Cricetinae ; Cricetulus ; Enzyme-Linked Immunosorbent Assay ; Epitopes ; immunology ; metabolism ; Hantaan virus ; immunology ; Microscopy, Confocal ; Oligopeptides ; immunology ; metabolism ; Peptide Library ; Vero Cells ; Viral Proteins ; immunology ; metabolism

OBJECTIVETo investigate the changes in rat cardiac function and myocardium ultrastructure in response to ouabain treatment.

METHODSTwenty-four male SD rats were randomized into two equal groups to receive daily intraperitoneal injection of ouabain or saline for 4 consecutive weeks, and their systolic blood pressure (SBP) was recorded weekly. After 4 weeks of injection, echocardiography was performed and the hemodynamic parameters were measured by invasive cardiac catheterization, and the changes in myocardium ultrastructure observed using transmission electron microscopy.

RESULTSAfter 4 weeks of ouabain injection, no significant changes in the mean SBP occurred in comparison with the saline group, but echocardiographic examination showed significant increases in the left ventricular end-diastolic and end-systolic diameters, septum thickness, posterior wall thickness, left ventricular mass and isovolumetric relaxation time but significantly lowered E/A ratio, ejection fraction and fractional shortening after ouabain treatment (P<0.05). Invasive monitoring revealed significant attenuation of the left ventricular developed pressure, rate of pressure development (+dp/dt) and rate of pressure decay (-dp/dt), and increment of the left ventricular end diastolic pressure. Myofibrillar fragmentation, swelling of the cardiac myocytes, absence of the Z line, increases of the mitochondria and collagen fibers were found in ouabain group by transmission electron microscopy.

CONCLUSIONOuabain can induce left ventricular enlargement, cardiac wall thickening, myocardial ultrastructural alterations, systolic and diastolic dysfunction in rats before blood pressure elevation is detected, indicating that ouabain can directly cause cardiac damage in rats.

Animals ; Echocardiography ; Heart ; drug effects ; physiopathology ; Male ; Microscopy, Electron, Transmission ; Myocardium ; pathology ; ultrastructure ; Ouabain ; pharmacology ; Random Allocation ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo introduce the application of reverse dorsal radial flap in reconstruction of distal segment defect of thumb.

METHODSFrom January 2004 to June 2007, 24 cases of soft tissue defect with exposed bone or tendon at the distal segment of thumb were involved. The flap size ranged from 2 cm x 2 cm to 3 cm x 4 cm.

RESULTSVenous congestion happened in 3 cases with one case of partial necrosis at the distal end of the flap. All the other flaps survived completely. The follow-up period was 6 - 24 months. The appearance and function of the thumbs were satisfactory. The 2-point discrimination was 7 - 11 mm (mean 9.3 mm).

CONCLUSIONSReverse dorsal radial flap is a good option for reconstruction of distal segment defect of thumb.

Adult ; Female ; Finger Injuries ; surgery ; Humans ; Male ; Middle Aged ; Skin Transplantation ; methods ; Soft Tissue Injuries ; surgery ; Surgical Flaps ; blood supply ; Thumb ; injuries ; Young Adult

Animals ; Graft Rejection ; immunology ; Liver Transplantation ; Male ; Rats ; Rats, Sprague-Dawley ; Rats, Wistar ; Sertoli Cells

OBJECTIVETo investigate the role of renal sympathetic nerves in renal sodium transport in ouabain-hypertensive rats (OHR).

METHODSSixteen male SD rats with sham renal denervation (Sham-RDNX) and 16 with renal denervation (RDNX) were randomly into normal control group and ouabain group to receive intraperitoneal injection of normal saline (1 ml/kg) and ouabain (27.8 µg/kg) once a day, respectively. Systolic blood pressure (SBP), heart rate and body weight were recorded weekly. Food consumption of the rats was determined twice a week. After a 4-week treatment, blood and 24 h urine samples were collected to measure the serum and urinary concentration of sodium, trace lithium and creatinine. Endogenous creatinine clearance rate (Ccr), fractional excretions of sodium (FENa), fractional excretions of lithium (FELi) and fractional reabsorption of sodium in the postproximal tubules (FDRNa) were calculated. Plasma renin activity was determined by radioimmunoassay. Norepinephrine was extracted from the renal tissue and assayed for norepinephrine content by HPLC.

RESULTSThe body weight, food intake and heart rate showed no significant difference among the 4 groups (P > 0.05). After 4 weeks, the SBP of control RDNX group (CDNX) was significantly lower than that of the control Sham-DNX group (Csham)(P < 0.05); the SBP of ouabain RDNX group (ODNX) was also significantly lower than that of ouabain Sham-DNX group (Osham) (P < 0.05); RNDX lowered SBP by about 10 mmHg in both ouabain groups and control groups. The SBP was significantly higher in Osham and ODNX groups than in the corresponding control groups (P < 0.01), also significantly higher in ODNX group than in Csham group (P < 0.01). Ccr showed no significant difference among the 4 groups(P > 0.05). FENa, FELi and FDRNa were significantly lower in ouabain groups than in the corresponding control groups (P < 0.05, P < 0.01, P < 0.05), but FENa, FELi and FDRNa of ODNX group were similar with those of Osham group (P > 0.05); FENa , FELi and FDRNa were similar between CDNX and Csham groups (P > 0.05). The plasma renin activity was comparable between the 4 groups (P > 0.05). Renal norepinephrine level was markedly reduced in RDNX group compared with that in Sham-RDNX group in both ouabain and control groups (P < 0.01).

CONCLUSIONThe increase of proximal tubule sodium reabsorption in OHR is not dependent on the renal sympathetic nerve.

Animals ; Hypertension ; chemically induced ; metabolism ; Kidney ; innervation ; Male ; Ouabain ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Sodium ; metabolism ; Sodium Channels ; metabolism ; Sympathetic Nervous System ; physiology

OBJECTIVETo evaluate the risk factors related to mortality in old patients with coronary heart disease after revascularization.

METHODSA total of 675 patients (498 males) with age >or= 70 years old who received revascularization during July 2003 to June 2004 and followed up > 30 days after discharge were included in this study. Clinical characteristics, death and major adverse cardiac and cerebral events (MACCE) during follow up were recorded.

RESULTSThe patients were followed up for a mean period of (754 +/- 355) days. 27 patients (4.0%) died and MACCE developed in 50 patients (7.4%) during follow up. Female and patients with anemia took a significantly higher risk of mortality (RR = 2.750, 95% CI 1.116 - 6.779, P = 0.028, RR = 0.385 95% CI 0.164 - 0.904, P = 0.028, respectively); Creatinine level is positively related to mortality rate. When comparing patients with Cr > 115 micromol/L and Cr > 177 micromol/L with patients with Cr < 115 micromol/L, the hazard rate was 2.963 and 10.785, respectively (95% CI 1.114 - 9.952, P = 0.035 and 95% CI 2.659 - 78.097, P = 0.000) after adjustment for other risk factors.

CONCLUSIONPreexisting anaemia (male Hb < 120 g/L, female Hb < 110 g/L), renal insufficiency (Cr > 115 micromol/L) and female gender were found to be independent risk factors for mortality in old patients with coronary heart disease post revascularization.

Aged ; Aged, 80 and over ; Coronary Disease ; mortality ; surgery ; Female ; Follow-Up Studies ; Humans ; Male ; Myocardial Revascularization ; Postoperative Period ; Prognosis ; Regression Analysis ; Risk Factors ; Sex Factors ; Survival Analysis