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Objective To perfect the purification method of recombinant fusion protein of Hespintor (rHespintor) for increasing the protein extraction efficiency,and to investigate its effects on the proliferation,migration and invasion of hepatoblastoma cell line HepG2.Methods In the recombinant protein extraction,the inclusion body washing process was added and the protein purification buffer system was changed.BAPNA was used as the substrate.The inhibitory effect tof purified rHespintor on trypsin hydrolysis was detected.The blank group served as the control group.The MTT test,cell scratch wound healing test and tumor cell invasion test were performed to detect the effect of rHespintor on growth of hepatoblastoma HepG2 cells and its effect.Results The urea gradient washing on the inclusion body protein could effectively remove the vast majority of impure proteins from the targeted protein.After one-step purification,the target protein rHespintor exhibited a high inhibition effect of trypsin hydrolysis,and the inhibitory effect was exhibited a dose-dependent manner.After acting on hepatoblastoma HepG2 cells with rHespintor,the cell proliferation ability was inhibited,the migration ability was reduced and the number of invaded cells were significantly decreased.Conclusion rHespintor can significantly inhibit the proliferation,migration and invasion of hepatoblastoma cell line HepG2 cells in vitro.
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Aim To evaluate the potency of anti-D. acutus venom IgY neutralizing the main activities of D. acutus venom.Methods After mixing the different a-mounts of IgY with snake venom and incubating togeth-er,the main activities of snake venom were assayed by biochemical methods.Results The in vitro assays in-dicated that anti-D.acutus venom IgY obviously neu-tralized the activities of PLA2 ,5′-nucleotidase,hyalu-ronidase,metalloprotease and serine proteinase (fi-brinogenase)in D.acutus venom.Mouse experiments showed that the ED50 value of IgY for mouse was 1 131.09 μg.Conclusion Anti-D.acutus venom IgY antibodies have good effects in neutralizing D.acutus venom without the toxicities themselves.
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Objective To investigate the expression and significance of Maspin and IKKα in nasosinusoidal mucosa of rats with fungal rhinosinusitis (FRS).Methods A total of 40 SD rats were used to establish the FRS model, and randomly divided into nasal obstruction group, FRS group, immunosuppressive group and invasive FRS group, 10 rats in each group.Another 10 normal rats were used as control group.Mice in the control group were fed with normal diet.In the nasal obstruction group, the mice had only hemostatic cotton stuffed in the nasal cavity and injection of 0.9% NaCl in the abdominal and nasal cavities.In the FRS group, the mice were injected Aspergillus fumigatus spore suspension into the nasal cavity and 0.9% NaCl i.p.The mice of the immunosuppressive group were given cyclophosphamide i.p.and 0.9% NaCl injection into the nasal cavity.The invasive FRS group was injected with cyclophosphamide i.p.and Aspergillus fumigatus spore suspension into the nasal cavity.The serum levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were measured by enzyme-linked immunosorbent assay (ELISA).The expression of Maspin and IKKα in nasosinusoidal mucosa was detected by immunohistochemical staining.The expression of Maspin mRNA and IKKα mRNA in the nasosinusoidal mucosa was detected by fluorescence quantitative PCR.Results The serum levels of IL-6 and TNF-α in different groups were significantly different (P 0.05).Theresult of immunohistochemical staining showed that the protein expression of Maspin in the FRS group and invasive FRS group was significantly lower than that in the control group, nasal obstruction group and immunosuppressive group, while the expression of IKKα protein was significantly higher than that of control group, nasal obstruction group and immunosuppressive group (P< 0.05).The protein expression of Maspin in the invasive FRS group was significantly lower than that in the FRS group, by contrast, the expression of IKKα protein was significantly higher (P< 0.05).The PCRresult revealed that the expression levels of Maspin and IKKα mRNA were (0.217 ± 0.013) and (0.193 ± 0.012), significantly lower than that in the control, obstruction and immunosuppressive groups [(0.309 ± 0.021), (0.302 ± 0.017), and (0.293 ± 0.02)] (P< 0.05), while the expressions level of IKKα mRNA were significantly higher [(0.319 ± 0.043), (0.384 ± 0.048) vs (0.169 ± 0.015), (0.171 ± 0.018), and (0.175 ± 0.019)] (P< 0.05).Conclusions Down-regulation of Maspin expression after IKKα activation is the main cause of the onset of FRS, which may also be one of the mechanisms of invasive FRS.
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Objective To observe the expression of estrogen receptors (ERαand ERβ) on gastric cancer cells and evaluate the effect of Tamoxifen(TAM) on the cell proliferation and expression of serine proteinase inhibitor 9(PI9) of gastric cancer cells . Methods PI9 positive expression(MNK45 ,SGC7901)and negative expression (BGC823)of gastric cancer cell lines were from pre‐liminary screened ,the expression of ERα and ERβ detected by immunofluorescence chemical method ,the cell proliferation and ex‐pression of PI9 were tested by CCK8 assay and reverse transcription‐PCR after intervention of TAM .Results ERαprotein expres‐sion was noted in MNK45 and SGC7901 ,ERβwas noted in BGC823 ,but the expression of ERαand ERβwere not appear to be obvi‐ous after the intervention of TAM .Tamoxifen could obviously inhibited cell proliferation of MNK45 and SGC7901 at concentration of 0 .1-100 .0 μmol/L ,the differences were statistically significant compared with negative control group (P<0 .05) ,but showed no dose‐dependent to the proliferation of BGC823 and MNK28 .After treating with TAM ,the expression of PI9 mRNA of SGC7901 (0 .402± 0 .020) and MNK45(0 .359 ± 0 .048) were obviously lower than that in the negatwe control group(P< 0 .05). Conclusion Tamoxifen could significantly inhibit the proliferation of PI9 positive expression than PI9 negative expression of gastric cancer cell lines ,and showed obviously dose‐dependent ,its role in inhibiting proliferation might closely related to immune tolerance improved by PI9 .
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Objective:To compare the expression and biological activity of glucosyl transferases (GTFs)of Streptococcus mutans (S.mutans)under normal outside environment between high temperature requirment serine proteinase A(HtrA)-deficient strains and high virulent strains isolated from children with high cario-susceptibility.Methods:The HtrA-deficient strains and high virulent strains of S.mutans were obtained by preliminary study.The strains were reanimated and incubated in BHI medium to exponential phase at tenth hour.The expression of gtfB,gtfC and gtfD were detected by real-time RT-PCR.The biological activity of the GTFs were detected by entong sulfuric acid method and Western Blot.Results:The expression of gtfs and GTFs in the HtrA-deficient strains was higher than those of high virulent strains,but the biological activity of the GTFs was lower.Conclusion:The HtrA gene plays an important regulatory role in the process of the GTFs expression of S.mutans isolated from children with high cario-susceptibility.
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Objective To explore the association between serum vaspin levels and the severity of the lower extremity vascular lesions in type 2 diabetes mellitus (T2DM) patients.Methods According to the lower extremity artery plaque,stenosis and intima-media membrane thickening severity score,92 cases of T2DM patients were divided into three groups:simple diabetes group (DM1 group) (32 cases),mild diabetic lower limb vascular lesion group (DM2 group) (33 cases),and moderately severe diabetic lower limb vascular lesion group (DM3 group) (27 cases).Twenty-six age-and gender-matched apparently healthy controls (control group) were recruited as well.Systolic blood pressure (SBP),ankle-brachial index (ABI),insulin resistance index (HOMA-IR),and other indicators were determined,serum vaspin was measured by enzyme-linked immunosorbent assay.Results After adjustment for age and gender,the DM1 group had a significantly higher serum vaspin level than control group (P ＜0.01),while for the DM2 and DM3 groups,serum vaspin levels were significantly lower than the DM1 group (P ＜ 0.01).Partial correlation analysis showed that serum vaspin was inversely associated with HOMA-IR (r =-0.461,P =0.001)and positively correlated with ABI (r =0.462,P =0.001).Logistic regression analysis demonstrated that SBP,ABI and fasting serum vaspin levels were significantly associated with the presence of the lower extremity vascular lesions in type 2 diabetes.Conclusions Serum vaspin levels were significantly elevated in simple type 2 diabetic patients while the reduction of its level might be associated with the formation of lower extremity vascular lesions in type 2 diabetic patients.
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Hepatitis C virus (HCV) genome is of high variation,which results in persistent infection of HCV and increases the incidence of liver cirrhosis and hepatocellular carcinoma.Following the successful paradigm established for HIV protease inhibitors,HCV NS3-4A serine protease has been selected as the main target for the development of small molecule antiviral agents.In this article,we review recent progress in the discovery and development of HCV NS3-4A protease inhibitors,and discuss their antiviral activities,pharmacokinetic properties,side effects and resistance profiles.
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Objective: To investigate the effects of TFPI-2 (tissue factor pathway inhibitor 2) on the growth and angiogenesis of subcutaneous tumor xenografts in nude mice established from human liver cancer cells. Methods: The Hep3B cells stably expressing TFPI-2 (Hep3B-TFPI-2 group) and the Hep3B cells transfected with empty vector PCDNA3.1 (Hep3B-V group) or without transfection (Hep3B-P group) were subcutaneously transplanted into nude mice respectively to generate subcutaneous tumor xenografts. The volume of tumor xenograft was measured every three days, and the growth curve of tumor xenograft was drawn when the subcutaneous tumor xenograft was visible. The nude mice were killed three weeks after transplant, the volume of tumor xenograft was measured, and the total RNAs and proteins in tumor xenografts were extracted. The mRNA and protein expressions of TFPI-2 and VEGF (vascular endothelial growth factor) in tumor xenografts were analyzed by RFQ-PCR (real-time fluorescence quantitative PCR) and Western blotting, respectively. The expression of TFPI-2 protein and the MVD (microvessel density) in tumor xenografts were observed by immunohistochemistry. Results: The eventual tumor volume of tumor xenografts in Hep3B-TFPI-2 group was apparently smaller than those in Hep3B-V group and Hep3B-P group (both P < 0.05). The expression of mRNA and abundance of protein of TFPI-2 in Hep3B-TFPI-2 group were significantly higher than those in the other two groups (P < 0.05); while the expression of mRNA and abundance of protein of VEGF in Hep3B-TFPI-2 group were apparently lower than those in the other two groups. Compared with Hep3B-V group and Hep3B-P group, the inhibitory rates of VEGF protein expression in Hep3B-TFPI-2 group were 19.8% and 23.5%, respectively (P < 0.05). The MVD in Hep3B-TFPI-2 group was apparently lower than those in the other two groups (P < 0.05). Conclusion: TFPI-2 can significantly inhibit the growth and angiogenesis of subcutaneous tumor xenografts in nude mice established from hepatocarcinoma Hep3B cells. Copyright © 2013 by TUMOR.
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Objective To investigate the expression and significance of Matriptase and HAI-1 protein in prostate cancer (CaP). Methods Specimens of 46 prostate cancers,20 benign prostate hyperplasias (BPH),10 high-grade intraepithelial neoplasias (PIN),and 10 normal prostates (NP) were used. Expressions of Matriptase and HAI-1 proteins in specimens were detected by SP of immunohistochemistry. The results were analyzed in relation to the clinicopathological data. Results The protein levels of Matriptase in CaP tissues were significantly higher than PIN tissues(Z＝-2.150,P＝0.032),and the expression of matriptase in CaP and PIN was higher than that in BPH and NP (Z＝-3.270,P＝0.001;Z＝-2.817,P＝0.005). No statistically significant difference was observed between BPH and NP group (Z＝-0.895,P＝0.325). A progressive increase in the protein levels of Matriptase was observed with increasing tumor grade (rs＝0.583,P＜0.01) and clinical stages（rs＝0.611,P＜0.01）in CaP specimens. The protein levels of HAI-1 in BPH and NP tissues were significantly higher than CaP and PIN tissues（Z＝-3.277,-3.315,P＜0.01）,the levels of HAI-1 in PIN were higher than CaP (Z＝-2.310,P＝0.020). No statistically significant difference was found between BPH and NP (Z＝-0.872,P＝0.330). A progressive decrease in the protein levels of HAI-1 was observed with increasing tumor grades（rs＝-0.634,P＜0.01） and clinical stages（rs＝-0.521,P＜0.01）. The expressions of Matriptase and HAI-1 in CaP tissues showed negative correlations（rs＝-0.712,-0.560,-0.465,respectively,P＜0.01）. Conclusions The abnormal expressions of Matriptase and HAI-1 proteins may be important events during the progression of CaP in humans. Matriptase and HAI-1 Protein may be used as parameters for assessing the malignancy and prognosis of CaP.
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Gyroxin, a thrombin-like enzyme isolated from Crotalus durissus terrificus venom and capable of converting fibrinogen into fibrin, presents coagulant and neurotoxic activities. The aim of the present study was to evaluate such coagulant and toxic properties. Gyroxin was isolated using only two chromatographic steps - namely gel filtration (Sephadex G-75) and affinity (Benzamidine Sepharose 6B) - resulting in a sample of high purity, as evaluated by RP-HPLC C2/C18 and electrophoretic analysis that showed a molecular mass of 30 kDa. Gyroxin hydrolyzed specific chromogenic substrates, which caused it to be classified as a serine proteinase and thrombin-like enzyme. It was stable from pH 5.5 to 8.5 and inhibited by Mn²+, Cu²+, PMSF and benzamidine. Human plasma coagulation was more efficient at pH 6.0. An in vivo toxicity test showed that only behavioral alterations occurred, with no barrel rotation. Gyroxin was not able to block neuromuscular contraction in vitro, which suggests that its action, at the studied concentrations, has no effect on the peripheral nervous system.
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Animals , Rats , Crotalid Venoms , Thrombin/isolation & purification , Thrombin/toxicityABSTRACT
Serine proteinase inhibitor9(P1-9),a charac-teristic member of serpins,has been identified as the only inhibitor of granzyme B(GrB).Accumulated evidence suggested that PI-9 inhibits GrB-induced apoptosis by blocking DNA fragmentation of target cell.Physiologically,PI-9 could protect cytotoxic lymphocytes from committing autolysis or fratricide,and play an important role in facilitating immunologic tolerance of immune-privileged sites.In addition,evidences in recent years suggest that PI-9 Was also involved in vailous pathologic processes,such as inflammation,trans plantation and immune tolerance of tumor.
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A serine proteinase with thrombin-like activity was isolated from the venom of the Central American pit viper Bothrops asper. Isolation was performed by a combination of affinity chromatography on aminobenzamidine-Sepharose and ion-exchange chromatography on DEAE-Sepharose. The enzyme accounts for approximately 0.13 percent of the venom dry weight and has a molecular mass of 32 kDa as determined by SDS-PAGE, and of 27 kDa as determined by MALDI-TOF mass spectrometry. Its partial amino acid sequence shows high identity with snake venom serine proteinases and a complete identity with a cDNA clone previously sequenced from this species. The N-terminal sequence of the enzyme is VIGGDECNINEHRSLVVLFXSSGFL CAGTLVQDEWVLTAANCDSKNFQ. The enzyme induces clotting of plasma (minimum coagulant dose = 4.1 µg) and fibrinogen (minimum coagulant dose = 4.2 µg) in vitro, and promotes defibrin(ogen)ation in vivo (minimum defibrin(ogen)ating dose = 1.0 µg). In addition, when injected intravenously in mice at doses of 5 and 10 µg, it induces a series of behavioral changes, i.e., loss of the righting reflex, opisthotonus, and intermittent rotations over the long axis of the body, which closely resemble the `gyroxin-like' effect induced by other thrombin-like enzymes from snake venoms.
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Animals , Mice , Blood Coagulation , Bothrops , Coagulants/isolation & purification , Crotalid Venoms/enzymology , Serine Endopeptidases/isolation & purification , Amino Acid Sequence , Antivenins/therapeutic use , Blood Coagulation/drug effects , Chromatography, Agarose , Chromatography, Ion Exchange , Costa Rica , Coagulants/administration & dosage , Coagulants/pharmacology , Drug Evaluation, Preclinical , Fibrinogen/metabolism , Serine Endopeptidases/chemistry , Serine Endopeptidases/genetics , Serine Endopeptidases/pharmacology , Snake Bites/physiopathology , Thrombin/chemistryABSTRACT
The pathogenic mechanism of granulomatous amebic encephalitis (GAE) and amebic keratitis (AK) by Acanthamoeba has yet to be clarified. Protease has been recognized to play an important role in the pathogenesis of GAE and AK. In the present study, we have compared specific activity and cytopathic effects (CPE) of purified 33 kDa serine proteinases from Acanthamoeba strains with different degree of virulence (A. healyi OC-3A, A. lugdunensis KA/E2, and A. castellanii Neff). Trophozoites of the 3 strains revealed different degrees of CPE on human corneal epithelial (HCE) cells. The effect was remarkably reduced by adding phenylmethylsulfonylfluoride (PMSF), a serine proteinase inhibitor. This result indicated that PMSF-susceptible proteinase is the main component causing cytopathy to HCE cells by Acanthamoeba. The purified 33 kDa serine proteinase showed strong activity toward HCE cells and extracellular matrix proteins. The purified proteinase from OC-3A, the most virulent strain, demonstrated the highest enzyme activity compared to KA/E2, an ocular isolate, and Neff, a soil isolate. Polyclonal antibodies against the purified 33 kDa serine proteinase inhibit almost completely the proteolytic activity of culture supernatant of Acanthamoeba. In line with these results, the 33 kDa serine proteinase is suggested to play an important role in pathogenesis and to be the main component of virulence factor of Acanthamoeba.
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Humans , Animals , Virulence Factors/isolation & purification , Virulence , Trophozoites/physiology , Substrate Specificity , Soil/parasitology , Serine Endopeptidases/isolation & purification , Epithelial Cells/parasitology , Encephalitis , Cornea/cytology , Cells, Cultured , Acanthamoeba castellanii/enzymology , Acanthamoeba Keratitis/parasitology , Acanthamoeba/classificationABSTRACT
Innate elastase inhibitors are known to be putatively involved in the regulation of tissue inflammation by inhibiting polymorphonuclear leukocyte (PMN) derived proteinases. The aim of this study was to evaluate affects of leukocyte elastase suppression and PMN infiltration on wound healing in mouse by administering the recombinant elastase inhibitor guamerin (rEIG) in two different wound models; 1) impaired pin-punctured dorsal mucosa of anterior tongue wound, 60 mice, treated with saline containing rEIG that were fed ad libitum and 2) stable linear excisional cutaneous wound, 40 mice, covered with fibrin sealant containing rEIG. The progress of healing was analyzed by histological methods. The tongue wounds treated with rEIG became edematous around the pin-punctured tongue wound, and influx of inflammatory cells and PMN into the underlying stromal tissue were seen rapidly after wounding and peaked between 2-4 days. Whereas the control mice showed almost no wheal formation in the pin-punctured wound, a far lesser levels of PMN infiltration, and almost complete wound closure in 4 days. In the other model, the liner excisional cutaneous wound treated with fibrin sealant containing rEIG showed early wound constriction, lesser degree of inflammatory cells influx, and complete reepithelialization in 4-5 days, whereas the wound of control mice with the fibrin sealant alone showed contrary delayed reepithelialization, greater degree of inflammatory cell infiltration, and consequencial formation of greater granulation tissue at wound site. Taken together, these data suggest paradoxical effects of rEIG on the wound healing where in the wound exposed to infiltrating milieu of microorganisms in the oral cavity, the rEIG aggravates the wound healing by interfering with other innate defensive factors and extended greater flux of PMNs to inflamed wound site, while in the wound enclosed by fibrin, the rEIG accelerated wound healing by inhibiting the inflammation-generated proteases and the acute inflammatory reaction.
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Animals , Female , Mice , Enzyme Inhibitors/pharmacology , Fibrin Tissue Adhesive/pharmacology , Invertebrate Hormones/analysis , Leukocyte Elastase/antagonists & inhibitors , Macrophages/immunology , Skin/drug effects , Tongue/drug effects , Wound Healing/drug effectsABSTRACT
This study was performed to investigate the biochemical properties of Acanthamoeba proteinase, its role in the pathogenesis of Acanthamoeba keratitis and the therapeutic effect of the homogenate of amniotic membrane as a proteinase inhibitors. Acanthamoeba castellanii isolated from the keratitis patient was cultured in PYG medium, in which the excretory and secretory products were analysed. The secretory proteinases of A. castellanii wre identified using in vitro azocasein assay, activity-PAGE, and various protein substrate degradation assays, and one of them was purified and characterized. The pruified secretory proteinase was a kind of serine proteinase. Its molecular weight was 105 kDa and optimal pH was 8.5. It was able to degrade the various protein substrates such as fibronectin, IgA, IgG, fibrinogen. The various proteinase ingibitors and the amniotic membrane homogenates were tested in vitro against the purified seirne proteinase. The amniotic membrane homegenates markedly showed the inhibitory effect against the enzyme activity and this inhibitory effect was also revealed in animal study. In vivo study, this purified proteinase was infected into 14 pigmented rabbit corneas, pretreated with steroids. The corneal lesions induced by both of the purified proteinase and A. castellanii, showed similar clinical findings each other, in which the stromal infiltration and opacity with epithelial defect was revealed. These corneal lesions were significantly inhibited without any side effects of the amniotic membrane homogenates. Conclusively, Acanthamoeba proteinase was closely associated with the pathogenesis of Acanthamoeba keratitis. This study provides a successful animal model of Acanthamoeba keratitis using pigmented rabbit. And the fact that Acanthamoeba-induced corneal lesions were inhibited by the amniotic membrane homogenate, suggested that the amniotic membrane homogenate have the ability of the serine protinase inhibition further investigative studies are also necessary.
Subject(s)
Animals , Humans , Acanthamoeba castellanii , Acanthamoeba Keratitis , Acanthamoeba , Amnion , Cornea , Fibrinogen , Fibronectins , Hydrogen-Ion Concentration , Immunoglobulin A , Immunoglobulin G , Keratitis , Models, Animal , Molecular Weight , Peptide Hydrolases , Serine , Serine Proteases , SteroidsABSTRACT
BACKGROUND: Trichophyton rubrum is the most common dermatophyte isolated from human and has ability to invade the tissues such as stratum comeum, nail and hair. The potential role of proteinases as virulence factors of F rMSrMm has been discussed at length. OBJECTIVE: As a first step towards assessing its virulence role, we report on the purification and characterization of proteinase from T. rubrum isolate culture filtrates. METHODS: An extracellular serine proteinase has been purified from culture filtrates of Trichophyton rubrum HP-9 by ultrafiltration, gel filtration chromatography, and affinity column chromatography. Azocoll and keratin azure were employed as the substrates of enzyme activities. Peak of proteolytic activity was analyzed by gelatin co-polymerized gel electrophoresis. RESULTS: The molecular weight of the purified enzyme was approximately exhibited to 14.0 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and molality of 14.0 kDa proteinase activity was 6.0 and 100mM, respectively. The activity was inhibited by serine proteinase inhibitor, phenylmethylsulfonyl fluoride (PMSF). The proteinase degraded gelatin, collagen type VI, and keratin from human epidermis but not hemoglobin. CONCLUSION: The 14,000 Mr extracellular serine proteinase purified from T. rubrum NP-9 culture filtrates has neutral pH optimum 6.0 and activities against gelatin, collagen type VI, and keratin.