转枯草芽孢杆菌纤溶酶(Bacillus subtilis fibrinolytic enzyme,BSFE)基因对烟草生长发育的影响

将枯草芽孢杆菌纤溶酶(Bacillussubtilisfibrinolyticenzyme,BSFE)基因及其信号肽序列转入烟草,转基因烟草表现出不同于野生型烟草的生长特性,如营养器官生长速率下降、叶片数目减少、叶片狭长、叶片宽度/叶片长度值降低以及植株矮小、节间缩短等。推测其与外源BSFE的蛋白水解活性有关。

Cloning and Expression of One Fibrinolytic Enzyme from Bacillus sp. zlw-2

The gene encoding fibrinolytic enzyme from Bacillus sp. zlw-2 was cloned and sequenced （accession no. EU734749）, which was 1146 bp, encoded 381 amino acids and had 99% homology with Nattokinase YF308 and NAT. The genes encoding pre-pro-fibrinolytic enzyme （including signal peptide, propeptide, and mature peptide） and fibrinolytic enzyme （including mature peptide） were cloned into pET28a vector respectively and then transformed into Escherichia coli BL21 （DE3）. The recombinant ofpre-pro-fibrinolytic enzyme showed enzyme activity of 183 U mL^-1, while no detectable enzyme activity could be found from the recombinant of the mature peptide.

Optimization of Douchi Fibrinolytic Enzyme Production by Statistical Experimental Methods

Thrombus disease, one of the common cardiovascular diseases, has attracted worldwide at- tention for its rising mortality and morbidity. Due to the distinct shortages of current fibrinolytic drugs, new fibrinolytic agents warrant investigation. In this study, 8 fibrinolytic enzyme-producing strains were isolated from Douchi--a traditional Chinese food, and strain XY-1 which produced the largest amount of the enzyme was chosen for the following experiments. The enzyme produced by strain XY-1 was named Douchi fibrinolytic enzyme （DFE）. We optimized the liquid culture medium of strain XY-1 for enzyme production using Plackett-Burman and Box-Behnken design. The predicted maximal DFE yield was 19.78 FU/mL with 11.4 g/L peptone, 0.5 g/L magnesium sulfate and 1 g/L sodium chloride. How- ever, we acquired maximal production of 21.33 FU/mL in actual experiments, equal to 107.84% of the theoretical value, and the yield had been increased by 79.55% as compared to the yield of un-optimized culture. It was demonstrated that the combined use of Plackett-Burman design and response surface methodology in fermentation optimization can effectively and rapidly increase DFE production.

Enzymatic properties of UFE,a novel marine fibrinolytic enzyme

A novel potent protease, Urechis unicinctus fibrinolytic enzyme （UFE）, was firstly discovered. The enzymatic properties of UFE were further investigated.As a low molecular mass protein,UFE appeared to be very stable to heat and pH.When temperature was below 50 ℃ ,the remnant enzyme activity remained almost unchanged, but when temperature was raised to 60 ℃ ,the remnant enzyme activity began to decrease rapidly. UFE was quite stable in the range of pH value from 3 to 12,especially in slightly alkaline pH value.Mn^2＋ ,Cu^2＋ and Fe^2＋ ions were activators of UFE, while Fe^3＋ and Ag^＋ ions were inhibitors of UFE.Fe^2＋ ion along with Fe^3＋ ion might regulate UFE activity in vivo. The optimum pH and temperature of UFE were about 8 and 50 ℃ ,respectively. Other characteristics of this enzyme were also studied. Systematic research results are significant when UFE is applied for medical and industrial purposes.

Interaction between Tb^(3 + )and Fibrinolytic Principle from Agkistrodon Acutus Venom

By using equilibrium dialysis, atomic absorption spectrometry, fluorescence titration and determination of fluorescence lifetime, it can be determined that each fibrinolytic principle(FP) molecule contains one Ca 2+ binding site and one Ca 2+ ion. The energy transfer between Tb 3+ and the Trp residue in FP was studied through fluorescence spectroscopy. Our studies show that the Ca 2+ ion in an FP molecule can be substituted by Tb 3+ ion. In FP molecule, the excition energy can be transfered from the Trp residue as an energy donor to Tb 3+ as an acceptor, substituted into FP and located near the Trp residue. The distance between Tb 3+ and the Trp residue, ～0 375 nm, was worked out with the experimental data and Forster theory.

Lessons learned from the TIMI trials in rescue interventions for elderly patients after failed fibrinolytic therapy:look beyond the TIMI flow

In ST-segment elevation myocardial infarction (STEMI), acute reperfusion of the infarct-related artery (IRA)is the main goal in the early minutes after the patient seeks medical attention. Fibrinolytic therapy (FT) and/or primary coronary intervention (PCI) were proven to be effective in opening the IRA.

FIBRINOLYTIC ACTIVITY OF PERITONEAL FLUID AND LUF IN INFERTILE WOMEN WITH ENDOMETRIOSIS

ＦＩＢＲＩＮＯＬＹＴＩＣＡＣＴＩＶＩＴＹＯＦＰＥＲＩＴＯＮＥＡＬ ＦＬＵＩＤＡＮＤＬＵＦＩＮＩＮＦＥＲＴＩＬＥ ＷＯＭＥＮ ＷＩＴＨ ＥＮＤＯＭＥＴＲＩＯＳＩＳＳｈｅｎＬｅｉ；（沈蕾），ＨｕａｎｇＺｈｕｌｉｎ（黄祝玲）（ＤｅｐａｒｔｍｅｎｔｏｆＯｂｓ...

EFFECTS OF EXERCISE ON THE FIBRINOLYTIC ACTIVITY INPATIENTS WITH UNSTABLE ANGINA PECTORIS

Objective The purpose of this study was to evaluate the fibrinolytic response to exercise in patients with unstable angina pectoris(UAP) compared with an age - matched control group. Methods We measuredtissure- type plasminogen activator (t- PA) activity and plasminogen activator inhibitor 1(PAI- 1) activitybefore and after treadmill exercise test in 20 healthy subjects and 25 patients with UAP. ResultsResting t - PA activities were similar between two groups, but resting PAI- 1 activity was higher in UAP groupthan in control. Although both groups showed significant increase in t- PA activity with exercise, post - exerciset - PA activity was significantly lower in patients with UAP than in control (0.96± 0.45IU/ml vs 1.89± 0.68I U/ml,P<0.01), Post - exercise PAI- 1 activity was still much higher in UAP group than in control (8.20±2.28A U/ml vs4.21± 0.68A U/ml, P<0.01). Conclusion There existed impaired fibrinolysis in patients with UAP not only at restbut also alter exercise loading.

Physicochemical Properties and Fibrinolytic Activity of Ginseng Powder Fermented with <i>Bacillus subtilis</i>Isolated from <i>Cheonggukjang</i>

In order to obtain all the properties of fermented ginseng, we fermented ginseng using Bacillus subtilis (B. subtilis) isolated from Cheonggukjang. A sterilized ginseng medium was made with 4-year-old ginseng powder and distilled water (300% ginseng powder [w/w]), and the ginseng was fermented by B. subtilis (1% ginseng medium) followed by incubation at 37°C for 3, 5, 7, and 10 days. The growth of B. subtilis in the ginseng medium significantly increased up to 9 log CFU/g, but no significant difference was observed after 3 days. As the fermentation progressed, the ginsenoside Rd and Rg+Rh1 contents increased by 255.3% - 322.5% and 165.6% - 228.6%, respectively, whereas the Rc, Re, and Rg1 contents decreased by 30.7% - 39.6%, 10.5% - 12.8%, and 16.2% - 16.6%, respectively. After 3 days of fermentation, a 6.25% - 7.12% viscous substance was produced;thereafter, the viscous substance was gradually reduced until it disappeared. The viscosity of the medium significantly decreased with a longer fermentation time. Fibrinolytic activity increased during 3 - 10 days of fermentation, indicating a relative activity of 85.0% - 100.0%.

Effect of Guhong injection combined with atyplase on vascular endothelial function and fibrinolytic system and related factors in patients with acute cerebral infarction

Objective:To explore the effect of Guhong Injection combined with atyplase on vascular endothelial function and fibrinolytic system and related factors in patients with acute cerebral infarction.Methods:To select 90 patients with acute cerebral infarction in our hospital,divided into control group(48 cases)and observation group(48 cases)randomly.Patients in the control group were treated with alteplase on the basis of routine symptomatic treatment,and the observation group was treated with Guhong injection on the basis of the control group.Before and after treatment,the serum levels of related factors were detected and compared between the two groups.Results:Before treatment,there were no significant differences in serum related factors(ET-1,PAO,H-FABP,VEGF,S100β,BDNF,CEC,Fibulin-5,vWF,P-selectin,t-PA,PAI-1)between the two groups;After treatment,the serum levels of ET-1,PAO,H-FABP,S100β,CEC,Fibulin-5,vWF,P-selectin,PAI-1 in the observation group were significantly lower than those in the control group,and the serum levels of VEGF,BDNF and t-PA were higher than those in the control group,there were significant differences between the two groups.Conclusion:Guhong injection was added to patients with acute cerebral infarction on the basis of routine symptomatic treatment and ateplase,could significantly improve the level of serum related factors,it was more conducive to the control of symptoms and rehabilitation of patients,the effect was definite,it was worth further study and application in clinic.

Production and Characterization of New Fibrinolytic Protease from Mucor subtillissimus UCP 1262 in Solid-State Fermentation

Fibrinolytic enzymes have received attention regarding their medicinal potential for thrombolytic diseases, a leading cause of morbidity and mortality worldwide. Various natural enzymes purified from animal, plant and microbial sources have been extensively studied. The aim of this work was to produce fibrinolytic protease by solid state fermentation using agro industrial substrates. Rhizopus arrhizus var. arrhizus UCP 1295 and Mucor subtillissimus UCP 1262 filamentous fungi species isolated from soil of Caatinga-PE, Brasil, were used as producer microorganisms. Wheat bran was shown to be the best substrate for the production of the enzyme and by using a 23 full factorial design the main effects and interactions of the quantity of the substrate wheat bran, moisture and temperature on the fibrinolytic enzyme production and protease were evaluated. The best results for fibrinolytic and protease activities, 144.58 U/mL and 48.33 U/mL, respectively, were obtained with Mucor subtillissimus UCP 1262 using as culture medium 3 g wheat bran, 50% moisture at a temperature of 25°C for 72 hours. The optimum temperature for the produced enzyme was 45°C and most of its original activity was retained after being subjected to 80°C for 120 min. The protease activity was enhanced by K+, Ca+ and Mn+;but with Cu+ there was an inhibition. The specificity to chromogenic substrate and the inhibition by PMSF indicates that it is a chymotrypsin-like serine protease. Presented results suggest that this enzyme produced by solid-state fermentation is an interesting alternative as a candidate for thrombolytic therapy.

Studies on a Novel Fibrinolytic Enzyme(Nattokinase)in the Vegetable Cheese Natto

The culture condition of bacillus strain and the extraction method of Nattokinase were reported. The fibrinolytic activity of nattokinese was appraised.

Discovery and Content Variation Analysis of Fibrinolytic Enzyme in Semen Sojae Praeparatum Fermentation

A strong fibrinolytic activity was demonstrated in the Semen Sojae Praeparatum(SSP), which is a famous traditional Chinese medicine. To study the activities and dynamic changes of fibrinolytic enzyme, standard fibrin plate was used to determine the fibrinolytic activity. For the first time fibrinolytic enzyme was found during the fermentation of SSP and the fibrinolytic activities of samples were shown to increase significantly over time. In the "yellow cladding" stage, the fibrinolytic activity was 619.75 IU/g. On day 6, 12 and 15 of the "secondary fermentation" stage, the fibrinolytic activity was 711.49 IU/g, 866.67 IU/g, 1 022.31 IU/g, respectively. The results indicate that fibrinolytic enzyme was generated during the fermentation of SSP and it displayed increasing activity which peaked at the "secondary fermentation" stage. The fibrinolytic enzyme was found to not only degrades fibrin directly, but also activate plasminogen to do so.

Coagulation and fibrinolytic activity in patients with acute cerebral infarction

Objective To measure the concentration of D-dimer (DD), tissue plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1) and plasminogen (PLG) activity in plasma and cerebrospinal fluid in patients with acute cerebral infarction and to investigate their clinical significance.Methods The concentrations of D-dimer, t-PA, and PAI-1 in plasma and cerebrospinal fluid in patients were measured by enzyme-linked immunosorbent assay (ELISA). The PLG biological activity was detected using the chromophore method. The results were compared with those of the controls.Results The concentrations of D-dimer, t-PA and PAI-1 in cerebrospinal fluid and plasma in patients with acute cerebral infarction were much higher than those of normal subjects (P<0.01). Conversely, the level of PLG activity was significantly lower in the patients than in the controls (P<0.01).Conclusion Hypercoagulability and secondary hyperfibrinolysis exist in patients with acute cerebral infarction.

Fibrinolytic Evaluation of Compounds Isolated from a Marine Fungus Stachybotrys Iongispora FG216

As a part of our continuing work to discover bioactive leading molecules from marine microorganism, ethyl acetate fraction of organic extract of the train Stachybotrys longispora FG216 showed fibrinolytic activity in our primary screen. The bioassay-guided purification of the active fractions resulted in isolation of a new isoindolone, FGFC2 （1） （FGFC2, Fungi fibrinolytic compound 2）, together with two known compounds, LL-Zl272β （2） and ergosterol （3）. The structure of compound 1 was elucidated by the spectral analysis of 1D （^1H, ^13C） NMR, 2D （COSY, HSQC, and HMBC） and ESI-MS. Three compounds were evaluated for fibrinolytic activities in vitro. Compared to FGFC1 （EC50=47 μmol/L） as a reference drug, compound 1 and ergosterol （3） showed moderate fibrinolytic activities in vitro with EC50 values of 108.16 and 156.30 μmol/L, respectively. LL-Z127213 （2） had no fibrinolytic activity.

Verticase:a Fibrinolytic Enzyme Produced by Verticillium sp,Tj33,an Endophyte of Trachelospermum jasminoides

Plant endophytes are among the most important resources of biologically active metabolites. Twenty-three endophyte strains residing in Trachelospermum jasminoides were cultivated in vitro with the cultures assayed for the fibrinolytic substance production. As a result, the culture of VerticUlium sp. Tj33 was shown to be the most active. A fibrinolytic enzyme designated as verticase was subsequently purified from the supernatant of Verticillium sp. culture broth by a combination of DEAE-52, Sephadex G-75 and hydrophobic column chromatographies. Verticase, with its molecular mass of 31 kDa and pl of 8.5, was demonstrated to be homogeneous by sodium dodecyl suIfate-polyacrylamide gel electrophoresis and isoelectric focusing electrophoresis. Verticase is an enzyme that hydrolyzes fibrin directly without activation of plaminogen. It was stable in a broad pH range from 4 through to 11 with the optimal reaction pH value and temperature shown to be around 9-10 and 50-60℃, respectively. The fibrinolytic activity of verticase was severely inhibited by phenylmethylsulfony fluoride, indicating that verticase was a serine protease.

Identification and Fibrinolytic Evaluation of an Isoindolone Derivative Isolated from a Rare Marine Fungus Stachybotrys Iongispora FG216

An isoindolone derivative, Fungi fibrinolytic compound （R）-2,5-bis（（2R,3R）-2-（（E）-4,8-dimethylnona-3,7-dien- 1-y1）-3,5-dihydroxy-2-methy--7-oxo-3,4,7,9-tetrahydr pyrano[2,3-e]isoindol-8（2H）-yl）pentanoic acid （FGFC1, Fungi fibrinolytic compound 1）, was isolated from a rare marine microorganism strain Stachybotrys longispora FG216. The structure of FGFC1 was elucidated by 1H NMR, 13C NMR, IR, and MS data; moreover, it was also evaluated for fibrinolytic activity in vitro and in vivo. The results showed that 0.1--0.4 mmol/L of FGFC1 could stimulate generation of plasmin activity （increased by 2.05--11.44 folds） by measuring Glu-plasminogen and Lys-plasminogen activation in vitro. The experiment of fluorescein isothiocyanate （FITC）-fibrinogen degradation indicated that the effect of FGFCI on fibrinolytic activity was mediated by plasminogen and scuPA. In addition, FGFC 1 （10 mg/kg） could dissolve most of pulmonary thrombus of Wistar rat in vivo. It is possible that FGFC 1 is a potential thrombolytic agent in the future.

Multi-isomorphous replacement phasing of the earthworm fibrinolytic enzyme component A from Eisenia fetida

Earthworm fibrinolytic enzyme component A (EFEa) from Eisenia fetida, a protein func-tioning not only as a direct fibrinolytic enzyme, but also as a plasminogen activator, has been crystallized in P212121 space group with 3 protein molecules per asymmetric unit. Four heavy atom derivatives were prepared using a mother liquor containing 1.4 mol·L-1 Li2SO4 and 0.1 mol·L-1 MOPS buffer (pH7.2) and used to solve the protein抯 diffraction phase. The heavy atom binding sites in the derivative crystals were determined using difference Patterson and difference Fourier methods and were refined in combination to yield the initial protein抯 structure phase at 0.25 nm resolution. The non-crystallographic symmetry relationship of the three independent protein mole-cules in the asymmetric unit was determined using the correlative heavy atom sites and used for the averaging of the initial electron density. As a result, the electron density was significantly im-proved, providing a solid foundation for subsequent structure determination.

Effect of reperfusion strategy on QT dispersion in patients with acute myocardial infarction:Impact on in-hospital arrhythmia

BACKGROUND Myocardial ischemia and ST-elevation myocardial infarction(STEMI)increase QT dispersion(QTD)and corrected QT dispersion(QTcD),and are also associated with ventricular arrhythmia.AIM To evaluate the effects of reperfusion strategy[primary percutaneous coronary intervention(PPCI)or fibrinolytic therapy]on QTD and QTcD in STEMI patients and assess the impact of the chosen strategy on the occurrence of in-hospital arrhythmia.METHODS This prospective,observational,multicenter study included 240 patients admitted with STEMI who were treated with either PPCI(group I)or fibrinolytic therapy(group II).QTD and QTcD were measured on admission and 24 hr after reperfusion,and patients were observed to detect in-hospital arrhythmia.RESULTS There were significant reductions in QTD and QTcD from admission to 24 hr in both group I and group II patients.QTD and QTcD were found to be shorter in group I patients at 24 hr than those in group II(53±19 msec vs 60±18 msec,P=0.005 and 60±21 msec vs 69+22 msec,P=0.003,respectively).The occurrence of in-hospital arrhythmia was significantly more frequent in group II than in group I(25 patients,20.8%vs 8 patients,6.7%,P=0.001).Furthermore,QTD and QTcD were higher in patients with in-hospital arrhythmia than those without(P=0.001 and P=0.02,respectively).CONCLUSION In STEMI patients,PPCI and fibrinolytic therapy effectively reduced QTD and QTcD,with a higher observed reduction using PPCI.PPCI was associated with a lower incidence of in-hospital arrhythmia than fibrinolytic therapy.In addition,QTD and QTcD were shorter in patients not experiencing in-hospital arrhythmia than those with arrhythmia.

Targeted thrombolysis strategies for neuroprotective effect

Stroke is usually treated by systemic thrombolytic therapy if the patient presents within an appropriate time window. There is also widespread interest in the development of thrombolytic agents that can be used in cases of delayed presentation. Current agents that can be used in cases of delayed presentation of nerve damage by thrombus. Current systemic thrombolytic therapy is associated with adverse effects such as fibrinogenolysis and bleeding. In an attempt to increase the efficacy, safety, and specificity of thrombolytic therapy, a number of targeted thrombolytic agents have been studied in recent years. This review focuses on the concepts underlying targeted thrombolytic therapy and describes recent drug developments in this field.