In order to improve the expression of recombinant human atrial natriuretic peptide (ANP), a new plasmid (pET28a(+)/ANP₃) containing 3 tandem ANP genes with lysine codon as the interval linker, was constructed. Target gene was transformed into Escherichia coli BL21 (DE3) and induced by IPTG, about 60% of the total-cell-protein was the target protein, His₆-ANP₃. After denaturation and refolding, it was digested by Endoproteinase Lys-C and Carboxypeptidase B (CPB) and then purified by a series of purification processes, about 16 mg purified ANP monomer could be obtained from one liter bacteria broth of shaking culture. Ultimately, the purity of protein was above 90% determined by UPLC and Tricine SDS-PAGE, its molecular weight was 3 080 Da according to LC-MS identification and it was proved to be equivalent to the reference product by ELISA. The use of tandem gene expression can provide a new possible model for the expression of other peptide drugs.
Atrial Natriuretic Factor ; biosynthesis ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; metabolism ; Gene Expression ; Humans ; Metalloendopeptidases ; Peptides ; Plasmids ; genetics ; Recombinant Fusion Proteins ; biosynthesis

Changes in microparticles (MP) from red blood cell (RBC) concentrates in the context of irradiation have not been investigated. The aim of this study was to evaluate how irradiation affects the number of MPs within transfusion components. Twenty RBC concentrates, within 14 days after donation, were exposed to gamma rays (dose rate: 25 cGy) from a cesium-137 irradiator. Flow cytometry was used to determine the numbers of MPs derived from RBC concentrates before and 24 hr after irradiation. The mean number of MPs (±standard deviation) in RBC concentrates was 21.9×10(9)/L (±22.7×10(9)/L), and the total number of MPs ranged from 2.6×10(9)/L to 96.9×10(9)/L. The mean number of MPs increased to 22.6×10(9)/L (±31.6×10(9)/L) after irradiation. Before irradiation, the CD41-positive and CD235a-positive MPs constituted 9.5% (1.0×10(9)/L) and 2.2% (263×10(6)/L) of total MPs, respectively. After irradiation, CD41-positive MPs increased to 12.1% (1.5×10(9)/L) (P=0.014), but the CD235a-positive MPs decreased to 2.0% (214×10(6)/L) of the total MPs (P=0.369). Irradiation increases the number of CD41-positive MPs within RBC concentrates, suggesting the irradiation of RBC concentrates could be associated with thrombotic risk of circulating blood through the numerical change.
Cell-Derived Microparticles/chemistry/*metabolism/radiation effects ; Erythrocytes/*cytology/radiation effects ; Flow Cytometry ; Gamma Rays ; Humans ; Membrane Glycoproteins/metabolism ; Metalloendopeptidases/metabolism ; Platelet Membrane Glycoprotein IIb/metabolism

The purpose of this study was to investigate the therapeutic effects of small hairpin RNA (shRNA) targeting endothelin-converting enzyme (ECE)-1 in monocrotaline (MCT)-induced pulmonary hypertensive rats. Ninty-four Sprague-Dawley rats were divided into three groups: control (n = 24), MCT (n = 35) and shRNA (n = 35). Four-week survival rate in the shRNA group was significantly increased compared to that in the MCT group. The shRNA group showed a significant improvement of right ventricular (RV) pressure compared with the MCT group. The MCT and shRNA groups also showed an increase in RV/(left ventricle + septum) ratio and lung/body weight. Plasma endothelin (ET)-1 concentrations in the shRNA group were lower than those in the MCT group. Medial wall thickness of pulmonary arterioles were increased after MCT injection and was significantly decreased in the shRNA group. The number of intra-acinar muscular pulmonary arteries was decreased in the shRNA group. The mRNA expressions of ET-1 and ET receptor A (ETA) were significantly decreased in the shRNA group in week 4. The protein levels of ETA were decreased in the shRNA group in week 2. The protein levels of tumor necrosis factor-alpha and vascular endothelial growth factor were decreased in the shRNA group in week 4. In conclusion, the gene silencing with lentiviral vector targeting ECE-1 could be effective against hemodynamic, histopathological and gene expression changes in pulmonary hypertension.
Animals ; Aspartic Acid Endopeptidases/*antagonists & inhibitors/blood/genetics ; Body Weight ; Heart Ventricles/physiopathology ; Hypertension, Pulmonary/chemically induced/*enzymology/mortality ; Lentivirus/genetics ; Lung/anatomy & histology/metabolism/pathology ; Male ; Metalloendopeptidases/*antagonists & inhibitors/blood/genetics ; Monocrotaline/toxicity ; Pulmonary Artery/drug effects/physiopathology ; RNA, Small Interfering/*metabolism ; Rats ; Rats, Sprague-Dawley ; Receptor, Endothelin A/genetics/metabolism ; Survival Rate ; Tumor Necrosis Factor-alpha/metabolism ; Vascular Endothelial Growth Factor A/metabolism

Atrial fibrillation (AF) is the most common sustained dysrhythmia in clinical practice. The bulk of evidence suggests that inflammatory processes, oxidative stress and matrix metalloproteinase are associated with development of AF. However, these agents may be involved in high mobility group box 1 protein (HMGB1). We hypothesized that HMGB1 may be a possible pathogenic link to AF. A growing body of evidence supports these hypotheses. First, the level of serum HMGB1 is significantly increased in patients with AF including paroxysmal and persistent AF. Second, HMGB1 has been identified as a new pro-inflammatory cytokine in cardiovascular diseases, along with tumor necrosis factor (TNF)-α, interleukin (IL)-6, and C-reactive protein, and there is cross-talk between HMGB1 and inflammatory cytokines. Third, oxidative stress is involved in the release of the pro-inflammatory cytokine, HMGB1, indicating there is cross-talk between oxidative stress and inflammation, and oxidative stress may reinforce the effect of inflammation on the pathogenesis of AF and inflammation may play a more important role in the pathogenesis of AF. Fourth, HMGB1 can promote matrix metalloproteinase-9 upregulation and activation. Fifth, HMGB1 receptors (receptor for advanced glycation end products, Toll-like receptor-2,4) may mediate the atrial structural remodeling or be up-regulated in patients with non-valvular AF. These results suggest that HMGB1 may participate in the pathogenesis of AF and provide a potential target for pharmacological interruption of AF.
Atrial Fibrillation ; metabolism ; HMGB1 Protein ; metabolism ; Humans ; Metalloendopeptidases ; metabolism ; Oxidative Stress ; physiology

Selection of suitable signal peptides is an important factor for efficient secretion of heterologous proteins. We defined structural fusion degree (SFD) as the compatibility degree of target proteins and signal peptides by a bioinformatics approach. We mathematically analyzed the interaction of fused signal peptides and adjacent residues of proteins, and proposed a mathematical model of extended signal region and the protein. SFD Features was extracted from this model to characterize the secretability of heterologous proteins. Simulation tests showed that SFD features can effectively discriminate high secretory proteins from poor ones in the host Bacillus subtilis. Results from this research will be useful in signal peptide selection and have a better guiding significance for the optimization of heterologous protein secretion.
Amino Acid Sequence ; Bacillus subtilis ; genetics ; metabolism ; Bacterial Proteins ; genetics ; metabolism ; Biotechnology ; methods ; Membrane Transport Proteins ; genetics ; metabolism ; Metalloendopeptidases ; genetics ; metabolism ; Molecular Sequence Data ; Protein Sorting Signals ; genetics ; Proteins ; secretion ; Recombinant Fusion Proteins ; genetics ; metabolism

OBJECTIVETo investigate 7 gene expression profile associated with inflammation and oxidative stress in vascular endothelium injure of rats with deficiency of vital energy or qi stagnation, and the effect of Tongxinluo on gene expression profile.

METHODThe model of vascular endothelium injury of rats with deficiency of vital energy or qi stagnation were established by using high L-methionine, with load-carrying swimming or being fastened, respectively. RT-PCR and SAGE database which is available in NCBI, were used to analyze the changes of 7 gene expression related with inflammation and oxidative stress in endothelium injure and the effect of Tongxinluo on the gene expression profile.

RESULTCompared with control group, the gene expression of inflammation related COX-1, COX-2, oxidative stress related iNOS, SOD and blood vessel vasomotion related eNOS, ECE, increased in deficiency of vital energy group (P < 0.05 or P < 0.01), and the gene expression decreased with Tongxinluo treatment (P < 0.05 or P < 0.01). The gene expression of COX-1, COX-2, iNOS and eNOS, ECE, increased (P < 0.01), but the gene expression of PCS and SOD decreased (P < 0.01), in qi stagnation group, and the disorder of gene expression improved with treatment of Tongxinluo (P < 0.01).

CONCLUSIONThe 7 gene expression related to vascular endothelium injure were not the same in rat with deficiency of vital energy or qi stagnation, and Tongxinluo could regulate the disorder of the gene expression, protecting vascular endothelium from injure.

Animals ; Aspartic Acid Endopeptidases ; genetics ; Cyclooxygenase 1 ; genetics ; Cyclooxygenase 2 ; genetics ; Drug Combinations ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Endothelin-Converting Enzymes ; Endothelium, Vascular ; injuries ; physiopathology ; Gene Expression ; drug effects ; Gene Expression Profiling ; Male ; Medicine, Chinese Traditional ; Metalloendopeptidases ; genetics ; Nitric Oxide Synthase Type II ; genetics ; Nitric Oxide Synthase Type III ; genetics ; metabolism ; Qi ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Superoxide Dismutase ; genetics

OBJECTIVETo investigate the expression of tumor stem cell marker CD133 and endothelin-converting enzymes (ECE) in non-small cell lung carcinoma (NSCLC) and their association with NSCLC lymphoid metastasis.

METHODSCD133 and ECE expressions was detected immunohistochemically in the specimens from 77 patients with NSCLC, and the association of CD133 and ECE expressions with the tumor size, histological type, differentiation, lymphoid metastasis, and prognosis of NSCLC was analyzed.

RESULTSThe positive expression rate of CD133 and ECE was 51.9% (40/77) and 45.5% (35/77) in these specimens, respectively. Both CD133 and ECE expressions were associated positively with lymphoid metastasis (r=0.246 and 0.339, P<0.05), and inversely with the survival time of the patients (P<0.05). CD133 and ECE expressions were not related to tumor size, histological type, and differentiation of the tumor (P>0.05). CD133 expression was associated positively with ECE expression in NSCLC (r=0.249, P<0.05).

CONCLUSIONCD133 and ECE expressions are associated with lymphoid metastasis and prognosis of NSCLC, and their overexpression often suggests unfavorable prognosis of NSCLC.

AC133 Antigen ; Adult ; Aged ; Antigens, CD ; biosynthesis ; Aspartic Acid Endopeptidases ; biosynthesis ; Carcinoma, Non-Small-Cell Lung ; metabolism ; pathology ; Endothelin-Converting Enzymes ; Female ; Glycoproteins ; biosynthesis ; Humans ; Immunohistochemistry ; Lung Neoplasms ; metabolism ; pathology ; Lymphatic Metastasis ; Male ; Metalloendopeptidases ; biosynthesis ; Middle Aged ; Peptides ; Prognosis

OBJECTIVETo analyze the effects of interaction between vascular endothelial cells and monocytes on the expression of matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase 2 (TIMP-2), as well as the regulation of pravastatin.

METHODSA co-cultured system of monocytes and endothelial cells was established through addition of THP-1 to human umbilical vein endothelial cells (HUVECs) in various rates. After 24 hours, the changes in activity and expression of MMP-2 and TIMP-2 in the co-culture system were studied by zymography and reverse zymography. The 1:1 co-culture system was selected and one control group (no pravastatin added) and experimental groups (with concentration of pravastatin being 0.1, 0.5 and 1.0 micromol/ml respectively) were studied. All groups were cultured for another 24 hours and analyzed in the same way.

RESULTSCompared to the single cultured HUVECs, the activity of proMMP-2 in the co-cultured system increased by 2.09, 2.46 and 2.07 folds respectively (number = 8, P < 0.01). There was also activated MMP-2 secretion in the co-culture system. The secretion of proMMP-2 and active MMP-2 in the 1:1 co-cultured system was most obvious. After pravastatin treatment, the activity of proMMP-2 and MMP-2, decreased significantly (number = 8, P < 0.01). MMP-2 secretion was completely suppressed after 1.0 micromol/ml pravastatin treatment. Reverse zymography revealed that, compared to the single culture HUVECs or THP-1, the secretion of TIMP-2 decreased in the co-cultured system, regardless of the ratio of mixture. However, pravastatin had no obvious effect on TIMP-2.

CONCLUSIONSInteraction between vascular endothelial cells and monocytes may contribute to the secretion and activation of MMP-2 and suppress secretion of TIMP-2. Pravastatin may inhibit the secretion and activation of MMP-2.

Anticholesteremic Agents ; pharmacology ; Cell Line, Tumor ; Cells, Cultured ; Coculture Techniques ; Endothelial Cells ; cytology ; metabolism ; Enzyme Precursors ; metabolism ; Gelatinases ; metabolism ; Humans ; Leukemia, Monocytic, Acute ; pathology ; Matrix Metalloproteinase 2 ; metabolism ; Metalloendopeptidases ; metabolism ; Monocytes ; cytology ; metabolism ; Pravastatin ; pharmacology ; Tissue Inhibitor of Metalloproteinase-2 ; metabolism ; Umbilical Veins ; cytology

In order to construct RGD-mSAK mutant with reduced immunogenicity, and identify its biological activity after purification, mSAK gene fragment was amplified by over-lapping extension PCR. Then the gene was inserted into the prokaryotic expression vector pBV220 with P(R)P(L) promoters after confirmed by DNA sequencing; the expression plasmid pBV220-RGD-mSAK was constructed, and then was transformed into E. coli. DH5alpha. After temperature induction, the mutant Staphylokinase was over-expressed and much of protein was in the supernate of lysate, which is over 50% of total protein in the host. The protein was isolated and purified in Q-Sepharose FF, Sephacryl S-200 and SP, high purity protein was obtained and its purity was over 98%. The thrombolysis activity of the RGD-mSAK protein is 1.68 x 10(5) u/mg by fibrin plate assay, which is slightly higher than that of the wild-type, and antiserum titers raised against this protein in guinea pigs were much lower than those of wild-type SAK, determined by ELISA. In anti-platelets aggregation assay in vitro, the RGD-mSAK protein has obvious inhibition activity of platelet aggregation in low concentration comparing to the control group and wild-type SAK group. So the RGD-mSAK protein is a low immunogenicity, bi-function molecular with both thrombolysis activity and anti-embolism activity. It provided the basis for further research of RGD-SAK.
Animals ; Base Sequence ; Escherichia coli ; genetics ; metabolism ; Guinea Pigs ; Metalloendopeptidases ; biosynthesis ; metabolism ; Molecular Sequence Data ; Mutant Proteins ; biosynthesis ; genetics ; Oligopeptides ; metabolism ; Platelet Aggregation ; drug effects ; Platelet Aggregation Inhibitors ; pharmacology ; Protein Engineering ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification

BACKGROUND/AIMS: Tumor angiogenesis, a major requirement for tumor growth and metastasis, is regulated by pro- and anti-angiogenic factors. Hepatocellular carcinoma (HCC) has become a common malignant tumor worldwide. It is characterized by a high vascularity. METHODS: We studied the immunohistochemical expression of angiostatin, vascular endothelial cell growth factor (VEGF), matrix metalloproteinase (MMP)-9 and MMP-12, and the relationship between these results and the microvessel density (MVD) in 48 HCC specimens. To determine whether HCC cells express angiostatin per se, we examined the expression of angiostatin, MMP-9 and MMP-12 by Western blotting in four HCC cell lines. RESULTS: Expression of angiostatin and MMP-12 (but not MMP-9) were strongly correlated with decreased MVD in HCCs (P=0.006, P=0.038, respectively). VEGF positive tumors showed a significantly higher MVD than VEGF negative tumors (P=0.01). We divided the 48 cases into the following four groups: group A, angiostatin (+), MMP-9 or -12 (+), and VEGF (-); group B, angiostatin (-) and VEGF (-); group C, angiostatin (+), MMP-9 or -12 (+), and VEGF (+); group D, angiostatin (-) and VEGF (+). There was a significant correlation with MVD among these groups (P<0.001). Angiostatin was detected by Western blotting in 2 out of 4 HCC cell lines and was associated with plasminogen and MMP expression. CONCLUSIONS: These results indicate that angiogenesis in HCC is a complex process involving multiple factors including angiostatin, VEGF, and MMP. Our results suggest that angiostatin is generated by MMP-mediated proteolysis of plasminogen in HCC cells.
Adult ; Aged ; Angiogenesis Inhibitors/analysis/physiology ; Angiostatins/analysis/physiology ; Carcinoma, Hepatocellular/*blood supply/chemistry ; English Abstract ; Female ; Gelatinase B/analysis/physiology ; Humans ; Liver Neoplasms/*blood supply/chemistry ; Male ; Metalloendopeptidases/analysis/physiology ; Middle Aged ; Neovascularization, Pathologic/metabolism/*physiopathology ; Vascular Endothelial Growth Factor A/analysis/physiology