The glycosylation heterogeneity of recombinant human pro-urokinase (pro-UK) was assessed using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). Firstly, the source of heterogeneity was determined by measuring the M_r of intact protein before and after N-deglycosylation. Glycosylation sites and the proportion of O-glycopeptides then were determined at the peptide level. Finally, the N-glycans were confirmed and quantified using the N-glycan profile. Results show that the structural heterogeneity of pro-UK is mainly caused by glycosylation. All T₁₈ were fucosylated, and 6.4% of S_138/139 was O-glycosylated with two kinds of oligosaccharides with a ratio of 6.0% and 0.4% respectively. All N₃₀₂ positions were N-glycosylated by more than ten types of glycans, among which A2F and A3F accounted for 80% of the total. The assessment of glycosylation heterogeneity of pro-UK will provide a reference for quality standardization.

OBJECTIVETo observe the effects of Shenqifuxin oral liquid(SQFXOL) on plasma kaliuretic peptide (KP), atrial natriuretic polypeptide(ANP), angiotension II (Ang II), endothelin(ET) and the left ventricular remodeling and the myocardial contractility and relaxation of left ventricle in experimental rats with heart failure(HF).

METHODThe SD rat model with HF was produced by constricting abdominal aorta. Hemodynamic parameters including maximum rate of intraventricular pressure rise (+dp/dtmax), left ventricular systolic pressure(LVSP), maximum velocity of contractile element shortening(Vmax), maximum rate of intraventricular pressure down(-dp/dtmax) and left ventricular end diastolic pressure(LVEDP) were measured by the method of the catheterization. Plasma concentrations of KP, ANP, Ang II and ET were determined by radioimmunoassays. The effects of treatment were evaluated by observing and comparing the changes of heart morphological structure, collagen element, heart weight/body weight ratio (HW/BW), left intraventricular area(LVA) and myocardial nuclei number (MNN) per square area.

RESULTIn high dose SQFXOL group, the LVSP, -dp/dtmax and Vmax were increased, while LVEDP was decreased, and plasma concentrations of KP, Ang II and ET were decreased. In comparision with those in model group, the difference was significant(P < 0.05 or P < 0.01). Though the +dp/dtmax and the level of ANP were decreased, the difference was insignificant(all P > 0.05). The collagen tissues around myocardial cells were reduced. HW/BW and LVA were lower, and MNN per square area was higher significantly (P < 0.05 or P < 0.01). The indices of +dp/dtmax in all of treatment groups and control group were not considerably different in comparison with those in model group. The levels of plasma ANP in middle dose group and low dose group were significantly lower than those in model group(all P < 0.01).

CONCLUSIONSQFXOL can reduce the plasma concentrations of KP, Ang II, ET, and ANP, improve the myocardial contractility and relaxation of left ventricular and inhibitate left ventricular remodeling in rats with HF.

Administration, Oral ; Angiotensin II ; blood ; Animals ; Astragalus membranaceus ; chemistry ; Atrial Natriuretic Factor ; blood ; Cardiotonic Agents ; administration & dosage ; pharmacology ; Drug Combinations ; Drugs, Chinese Herbal ; administration & dosage ; isolation & purification ; pharmacology ; Endothelins ; blood ; Heart Failure ; blood ; physiopathology ; Male ; Myocardial Contraction ; drug effects ; Ophiopogon ; chemistry ; Panax ; chemistry ; Plants, Medicinal ; chemistry ; Protein Precursors ; blood ; Rats ; Rats, Sprague-Dawley ; Ventricular Function, Left ; Ventricular Remodeling ; drug effects

OBJECTIVETo investigate the clinical effect of manshuailing oral liquid on patients with congestive heart failure of type heart and kidney Yang deficiency.

METHOD90 patients of heart failure were randomly divided into 2 groups. 45 cases in the routine treatment group (RT) received general therapy including diuretics and digitalis, and 45 cases in the Chinese herb medicine group (CH) were treated basically with the above medicine, with additional manshuailing oral liquid. The clinical effect was summarized 6 weeks after treatment.

RESULTTotal effect rate was 82.2% and 62.2% in CH and RTgroup respectively. Compared with pretreatment, heart function including stroke volume (SV), stroke volume index (SVI), cardiac index (CI), shorten rate of left ventricular short axe (deltaD%), distance of inter-ventricular septal to mitral valve (EPSS) were all improved significantly in both groups (P < 0.05 or P < 0.01), and with even better effects in the CH group than the RT group (P < 0.05 or P < 0.01), except the SV.

CONCLUSIONManshuailing oral liquid can alleviate clinical symptom, decreased EPSS, increase deltaD% and improve heart function.

Administration, Oral ; Adult ; Aged ; Cardiotonic Agents ; therapeutic use ; Combined Modality Therapy ; Diagnosis, Differential ; Digoxin ; therapeutic use ; Drug Combinations ; Drugs, Chinese Herbal ; administration & dosage ; isolation & purification ; therapeutic use ; Female ; Heart Failure ; drug therapy ; physiopathology ; Heart Function Tests ; Humans ; Hydrochlorothiazide ; therapeutic use ; Isosorbide Dinitrate ; therapeutic use ; Male ; Medicine, Chinese Traditional ; Middle Aged ; Phytotherapy ; Plants, Medicinal ; chemistry ; Sodium Chloride Symporter Inhibitors ; therapeutic use ; Vasodilator Agents ; therapeutic use ; Yang Deficiency ; drug therapy

To establish methods and requirements for quality control of rhLFA3-IgG1, biological potency of rhLFA3-IgG1 was determined by CD2 molecule competitive binding assay on Jurkat cell surface. Purity of rhLFA3-IgG1 was analyzed by SEC-HPLC and IEC-HPLC. Peptide mapping was preformed by tryptic digestion and RP-HPLC after sample reduced and carboxymethylation by DTT and indoacetic acid, respectively. CHO host cell protein and Protein A residual were detected by ELISA separately. The quality control methods and requirements, such as biological potency, the physical-chemical characteristic of rhLFA3-IgG1 had been established. The methods and requirements for quality control of rhLFA3-IgG1 showed advantages of assuring the products safety and efficacy, which can be used for routine quality control of rhLFA3-IgG1.
Binding, Competitive ; Biotechnology ; methods ; CD2 Antigens ; metabolism ; CD58 Antigens ; biosynthesis ; chemistry ; Chromatography, High Pressure Liquid ; Humans ; Immunoglobulin G ; biosynthesis ; chemistry ; Jurkat Cells ; Molecular Weight ; Peptide Mapping ; Quality Control ; Recombinant Fusion Proteins ; biosynthesis ; chemistry

To establish a detection method of oncolytic adenovirus/p53 and standard of quality control, human telomerase reverse transcriptase (hTERT) promoter, CMV fusion promoter containing hypoxia reaction element (HRE) and p53 gene were identified by vector DNA restriction enzyme digestion and PCR analysis. The result conformed that all modified regions were in consistent with theoretical ones. Particle number was 2.0 x 10(11) mL(-1) determined by UV (A260). Infectious titer was 5.0 x 10(10) IU mL(-1) analyzed by TCID50. In vitro p53 gene expression in human lung cancer cell H1299 was determined by ELISA, and A450 ratio of nucleoprotein in virus infection group to control group was 5.2. Antitumor potency was evaluated by cytotoxicity assay using human lung cancer cell A549, and the MOI(IC50) of this gene therapy preparation was 1.0. The tumor cells targeted replication ability of recombinant virus was determined by TCID50 titer ratio of filial generation virus between human lung cancer cell A549 and human diploid epidermal fibrolast BJ cells after infected by virus with same MOI. TCID50 titer ratio of tumor cell infection group to normal cell infection control group was 398. The IE-HPLC purity of virus was 99.5%. There was less than 1 copy of wild type adenovirus within 1 x 10(7) VP recombinant virus. Other quality control items were complied with corresponding requirements in the guidance for human somatic cell therapy and gene therapy and Chinese pharmacopeia volume III. The detection method of oncolytic adenovirus/p53 was successfully established for quality control standard. The study also provided reference for quality control of other oncolytic viral vector products.
Adenoviridae ; genetics ; metabolism ; physiology ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Genes, p53 ; Genetic Therapy ; Genetic Vectors ; Humans ; Neoplasms ; metabolism ; pathology ; virology ; Oncolytic Viruses ; genetics ; metabolism ; physiology ; Quality Control ; Recombinant Fusion Proteins ; genetics ; metabolism ; Transfection ; Virus Replication

OBJECTIVETo investigate the effects of simvastatin (Sim) and the interference by mevalonate (MVA) against its effect on DNA synthesis in rat cardiac fibroblasts (CFs).

METHODSCFs were isolated from neonatal SD rats by trypsin digestion and growth-arrested CFs were stimulated with Sim and/or MVA at varied concentrations for different time lengths, and the DNA synthesis in the cells was measured by (3)H-thymidine ((3)H-TdR) incorporation assay.

RESULTSSim decreased (3)H-TdR incorporation in the CFs in a concentration-dependent manner, and (3)H-TdR incorporation was significantly lower in cells treated with 1 x 10(-6) and 1 x 10(-5) mol/L Sim (1,175+/-202.66 and 771+/-164.86 cpm/2000 cells, respectively) than in the control cells (1,608+/-204.32 cpm/2000 cells, P<0.01). As the treatment time with 1 x 10(-5) mol/L Sim prolonged (for 6, 12, 18, 24, 36, 42, and 48 h), (3)H-TdR incorporation in CFs decreased gradually, showing an obvious inverse correlation with the treatment time (r=-919, P<0.01). (3)H-TdR incorporation in cells treated with 1 x 10(-6) to 1 x 10(-3) mol/L MVA and 1 x 10(-5) mol/L Sim rose steadily as MVA concentration increased. A significant difference in the incorporation was found between cells treated with both 1 x 10(-4)/1 x 10(-3) mol/L MVA and 1 x 10(-5) mol/L Sim (1,612+/-308.57 and 1,995+/-353.83 cpm/2000 cells, respectively) and the cells with 1 x 10(-5) mol/L Sim treatment alone (P<0.01); difference was also noted between cells treated with 1 x 10(-5) mol/L MVA and the control cells (P<0.05), but treatment with 1 x 10(-6) mol/L MVA did not produce much difference in comparison with the control cells (P>0.05) With the increase of treatment time (for 6, 12, 18, 24, 36, 42, 48 h), 1 x 10(-3) mol/L MVA caused steady increase in (3)H-TdR incorporation in the CFs, showing a significant positive correlation with the treatment time (r=0.968, P<0.01).

CONCLUSIONSim can decrease DNA synthesis in rat CFs and postpone the occurrence of myocardial fibrosis, which can be reversed by MVA.

Animals ; Animals, Newborn ; Cells, Cultured ; DNA ; biosynthesis ; Dose-Response Relationship, Drug ; Female ; Fibroblasts ; cytology ; drug effects ; metabolism ; Fibrosis ; prevention & control ; Hypolipidemic Agents ; pharmacology ; Male ; Mevalonic Acid ; pharmacology ; Myocardium ; metabolism ; pathology ; Myocytes, Cardiac ; cytology ; drug effects ; metabolism ; Rats ; Rats, Sprague-Dawley ; Simvastatin ; pharmacology ; Time Factors

The primary structure of TNK-tissue plasminogen activator (TNK-tPA) was characterized using liquid chromatography-mass spectrometry (LC-MS). Firstly, the molecular mass of deglycosylated protein was measured. Then peptide mass mapping and MS/MS of the reduced, alkylated and trypsin-digested sample were tested and analyzed so as to verify its amino acid sequence and identify post-translational modifications. Results show that the amino acid sequence was consistent with designed structure; about 5% of M207 was oxidized; T61 was fucosylated with -80% occupancy; N103, N448 and N184 (-15% occupancy) were glycosylated with complex-type oligosaccharides. LC-MS coupled with proper sample pretreatment is approved to be a rapid and powerful approach to characterize the primary structure of TNK-tPA.
Amino Acid Sequence ; Chromatography, Liquid ; Glycosylation ; Mass Spectrometry ; Molecular Weight ; Protein Processing, Post-Translational ; Tissue Plasminogen Activator ; chemistry

Objective To study the expression of protein 94 (GRP94) and C/EBP-homologous protein (CHOP) in myocardial tissue of hypertensive rats and to investigate the effects of amlodipine on endoplasm retieulum stress ( ERS) and ventricular hypertrophy in abdominal aortic banded rats .Methods One hundred and twenty adult male SD rats with criteria were divided randomly into three groups:sham-operated group , abdominal aortic banding ( AAB) and AAB treated with amlodipine (AAB+Aml)groups(n=40).In sham-operated rats, the abdominal aorta was isola-ted but not constricted , while the abdominal aorta were constricted in AAB rats .The AAB+Aml group was treated by abdominal aortic constriction and treated with amlodipine [10 mg/(kg· d)].According to the time of surgery, each group was further divided into 2, 4 and 8-week postoperative subgroups ( n=6 ) .Mean arterial pressure (MAP), the 1eft ventricular mass (LVM) and body mass (BM) were measured and (LVM/BM) was calculated. The morphology of cardiomyocytes was observed by HE staining .The protein level of GRP 94 and CHOP was ana-lyzedbyimmunohistochemistryandwesternblot.Results 1)Withtimeaftersurgery,MAPandLVM/BWof AAB group increased gradually , and they were obviously higher than the sham operation group [ MAP: 2 weeks (144 ±10)vs(118 ±9), 4 weeks (163±8)vs(120±7), 8 weeks(177±10)vs(120±6)mmHg;LVW/BW:2 weeks (2.21±0.17) vs (1.91±0.12), 4 weeks (2.45±0.16) vs (2.01±0.14), 8 weeks (2.68±0.15) vs ( 2.05 ±0.09 ) mmHg;( P<0.05 ) ] .The cardiomyocytes in AAB group were hypertrophic as compared to the sham group.The expression of GRP94 in AAB group increased significantly at 2 weeks post-operation, and reached peak level at 4 weeks after the surgery and was on the decline thereafter .The expression of CHOP and GRP94 in AAB rats were significantly higher than sham group, and reached the peak at the 8 weeks after surger-y.2)Treatment with amlodipine significantly reduced MAP and LVM/BW in AAB rats[(MAP:2 weeks (126± 6) vs (144±10), 4 weeks (125±8) vs (163±8), 8 weeks (128±5) vs (177±10)mmHg;LVM/BW:2 weeks ( 1.94 ±0.15 ) vs ( 2.21 ±0.17 ) , 4 weeks ( 2.13 ±0.08 ) vs ( 2.45 ±0.16 ) , 8 weeks ( 2.18 ±0.10 ) vs ( 2.68 ± 0.15)mg/g;(P<0.05)].The myocardial hypertrophic was alleviated in AAB+Aml group.The level of GRP94 and CHOP in AAB+Aml group was lower than those in AAB rats ( all P<0.05 ) .Conclusions Endoplasmic reticulum stress is involved in the ventricular remodeling caused by hypertension , and use of amlodipine can reduce ventricu-lar hypertrophy in hypertension models possibly by inhibiting endoplasmic reticulum stress via down -regulation of GRP94 and CHOP .

OBJECTIVE We want to investigate the mechanism of organophosphate- induced delayed neuropathy (OPIDN) and find appropriate therapeutic medicine. OPIDN, often leads to pares?thesias, ataxia and paralysis, occurs in the late-stage of acute poisoning or after repeated exposures to organophosphate (OP) insecticides or nerve agents, and may contribute to the Gulf War Syndrome. METHODS FDSS Ca2 +-influx assays, single-cell calcium imaging and patch-clamp electrophysiology were the major testing techniques. Transfected HEK293 cells and dorsal root ganglion (DRG) neurons were used to evaluate the effects of compounds. Wild type and trpa1 knockout mice and adult hyline brown hens were used to evaluate the neuropathological damages caused by the OPs. Transmission electron microscopy imaging was used to observe the nerve injuries ultrastructurally. High-throughput screen for TRPA1 inhibitors was accomplished by Ion Works Barracuda (IWB) automated electrophysiology assay. RESULTS TRPA1 (Transient receptor potential cation channel, member A1) channel mediates OPIDN. A variety of OPs, exemplified by malathion, activates TRPA1 but not other neuronal TRP channels. Malathion increases the intracellular calcium levels and upregulates the excitability of mouse DRG neurons in vitro. Mice with repeated exposures to malathion also develop local tissue nerve injuries and pain-related behaviors, which resembles the early symptoms of OPIDN. Both the neuropathological changes and the nocifensive behaviors can be attenuated by treatment of TRPA1 antagonist HC030031 or abolished by knockout of Trpa1 gene. In the classic hens OPIDN model, malathion causes nerve injuries and ataxia to a similar level as the positive inducer tri-ortho-cresyl phosphate (TOCP), which also activates TRPA1 channel. Treatment with HC030031 reduces the damages caused by malathion or TOCP. Duloxetine and Ketotifen, two commercially available drugs exhibiting TRPA1 inhibitory activity, show neuroprotective effects against OPIDN and might be used in emergency situations. CONCLUSION TRPA1 is the major mediator of OPIDN and targeting TRPA1 is an effective way for the treatment of OPIDN.

The aim of current study was to detect intron 22 inversion of factor VIII gene in severe hemophilia A (HA) patients and screen the carriers of the gene inversion. Fifty-five cases of severe HA were involved and factor VIII gene inversion was detected and identified by long distance-PCR (LD-PCR) and 0.6% agarose gel electrophoresis. The 11 kb and 12 kb bands indicate the factor VIII gene inversion and non-inversion, respectively. Occurring of both 11 kb and 12 kb bands indicates a carrier of the inversion. The results showed that factor VIII gene inversion existed in 22 out of 55 cases, which accounted for about 40% of total detected patients. Five carriers of factor VIII gene inversion were diagnosed from the members in 15 families. In conclusion, LD-PCR assay is a simple, rapid and accurate method for detection of factor VIII gene inversion, and this approach is helpful in screening, carrier testing, and prenatal diagnosis of severe hemophilia A.
Adolescent ; Adult ; Antigens ; analysis ; Child ; Child, Preschool ; Chromosome Inversion ; Factor VIII ; genetics ; Hemophilia A ; blood ; genetics ; Humans ; Infant ; Male ; Polymerase Chain Reaction ; methods ; von Willebrand Factor ; immunology