酶法有限水解对大豆分离蛋白乳化性能的影响

以蛋白酶Alcalase作用于大豆分离蛋白 ,分析了有限水解作用对产物乳化性能的影响。在实验条件下 ,当水解度 <6.0时 ,水解产物的乳化性能随其溶解性的增加而改善。在广泛pH范围内 ,乳化能力、乳化活性及等电点附近的酸凝性均比未处理前有明显提高。在pH >5.5时 ,乳化稳定性比对比样品降低幅度约高 2 .5～ 5.0倍 ;而在pH≤ 5.5时 ,则无明显改变。

6 Kinds of Isozymes after Long-term Subculture of Emmenopterys henryi Oliv.

[Objective] The aim was to study the changes of zymography in 6 kinds of isozymes after long-term subculture of Emmenopterys henryi Oliv.. [Method] Non-denaturing polyacrylamide gel electrophoresis was used to analyze isozyme patterns such as esterase (EST),acid phosphatase (ACP),ATP enzyme (ATPase),amylase (AMY),superoxide dismutase (SOD) and peroxidase (POD) in long-term subculture callus of Emmenopterys henryi Oliv. [Result] The research showed that there were differences among the 6 kinds of isozymes in embryogenic callus and non-embryogenic callus of Emmenopterys henryi Oliv.,and both levels could be taken as the basis for identification,the EST,ACP,and POD of non-embryogenic callus were significantly higher than embryogenic callus. The browning of non-embryogenic callus was non-level in the AMY,SOD and POD isozymes when was compared with normal non-embryogenic callus,while the EST,ACP and ATPase isozymes decreased; When the browning of embryogenic callus was contrasted with normal embryogenic callus,EST isozyme increased and the other 5 kinds of enzymes decreased. [Conclusion] The study provided theoretical basis for research morphological difference and browning of long-term tissue culture of Emmenopterys henryi Oliv..

PEGylation of Hirudin and Analysis of Its Antithrombin Activity in vitro

Hirudin is the most anticoagulant drug found in nature, but its short serum half-life significantly inhibits its clinical anpplication. The PEGvlation of hirudin, the most promising anticoagulant drug, was performed in this paper. The optimal reaction conditions for PEG ylated hirudin were investigated, wh.en the PEGylation react, on.wasconducted under 4℃ after 10h, in the borate buffer at pH 8.5 .with the molar ratio 230 ： 1 of PEG to hirudin, a higher modification extent was achieved. Finally, the bioactivity of PEGylated hirudin was measured in vitro.Compared with unmodified hirudin, 26% of anti-thrombin activity was retained.

Comparison of ultrasound-guided thrombin injection and compression repair in treatment of iatrogenic femoral arterial pseudoaneurysms

Objective ：To retrospectively compare the efficacy and safety of ultrasound-guided thrombin injection （UGTI） with ultrasound-guided compression repair （UGCR） in patients with postcatheterizational femoral arterial pseudoaneurysms （PSA）. Methods： Thirty patients of this iatrogenic PSA [8 males, 22 females, average age （66.5±5.2） years] in our institution from 1997 to 2004 were retrospectively analyzed. Among them, 11 patients were treated with UGCR, 2 under continuous uhrasonographic （US） guidance and 9 under the guidance of femoral arterial bruit auscultation and dorsalis pedis artery palpation. Because UGCR was failed in 5 patients, consecutively 24 patients were treated with UGTI. Swine thrombin solution at a concentration of 200 U/ml was injected percutaneously using 22-25 gauge needles under color Doppler US. Demographics, clinical variables, pseudoaneurysm characteristics, and results of the 2 groups were compared by using Fisher＇s exact test and Student＇s t test. Results： The initial success rate of UGCR was 36.4% （4/11） and the overall success rate was 45.5% （5/11）. Tenor 11 patients suffered from local pain during the compression, but there was no any complication in UGTI group. The av- erage dose of injected thrombin was （180±82） U for PSA of a single loculus and （315±150） U for multi- loculated PSA. The initial success rate of UGTI was 89.5% （17/19） and the overall success rate was 100% （24/24）. Conclusion：UGTI offers a safe, quick and effective means of definitively treating femoral pseudoaneurysms and seems superior to UGCR. The amount of thrombin applied on our people seems smaller compared with others＇ work.

Digestive enzyme activity and mRNA level of trypsin in embryonic redclaw crayfish, Cherax quadricarnatus

Abstract The digestive enzyme activity and mRNA level of trypsin during the embryonic development of Cherax quadricarinatus were analyzed using biochemical and Fluorogenic Quantitative PCR （FQ-PCR） methods. The results show that the activities of trypsin and chymotrypsin had two different change patterns. Trypsin specific activity increased rapidly in the early stages of development and still remained high in preparation for the hatch stage. However, chymotrypsin activity peaked in stage 4 of embryonic development and decreased significantly in the last stage. The mRNA level of trypsin was elevated in all stages and two peak values were observed in stages 2 and 5 respectively. The results indicate that trypsin is very important for the utilization of the yolk during embryonic development and for the assimilation of dietary protein for larvae. The gene of trypsin is probably regulated at transcriptional level. The mRNA levels of trypsin can reflect not only trypsin activity, but also the regulatory mechanism for expression of trypsin gene to a certain degree.

Effects of fused hirudin on activity of thrombin and function of platelets

Objective: To investigate whether fused hirudin peptide has both antithrombin and antiplatelet functions. Methods: The core region of fused hirudin was the C-terminal tail of hirudin(hirudin_ 53-64),which could bind to the anion binding exosite (ABE) of thrombin.Arg-Pro-Pro-Gly-Phe(RPPGF) amino acid sequence,a metabolite of bradykinin,was added to the N-terminus of hirudin_ 53-64.It bound to the active site of thrombin.Additionally,Arg-Gly-Asp(RGD)amino acid sequence,an inibitor of glycoprotein Ⅱb/Ⅲa( GP Ⅱb/Ⅲa) receptor,was linked to C-terminus of hirudin_ 53-64.This 26-animo acid-fused hirudin peptide was artificially synthesized,purified and analysed. Results: Fused hirudin peptide significantly lengthened the activated partial thromboplastin time(APTT),thrombin time(TT)and prothrombin time(PT) and inhibited the amidolytic activity of thrombin.The ADP-induced platelet aggregation was markedly inhibited by fused hirudin peptide. Conclusion: Fused hirudin peptide has activity of antithrombin as well as antiplatelet.Therefore bifunctional anticoagulation peptide has capacity to target various components of haemostatic process and may become more powerful antithrombosis agent.

Liver-protecting effects of omega-3 fish oil lipid emulsion in liver transplantation

AIM:To investigate the liver-protecting effect of parenteral nutrition(PN) support with omega-3 fatty acids in a randomized controlled clinical trial.METHODS:Sixty-six patients with the diagnosis of end-stage liver disease or hepatic cellular carcinoma were admitted to the Affiliated Drum Tower Hospital,Nanjing University,China for orthotopic liver transplantation.The patients were randomly divided into two groups:PN group(n = 33) and polyunsaturated fatty acid(PUFA) group(n = 33).All patients received isocaloric and isonitrogenous PN for seven days after surgery,and in PUFA group omega-3 fish oil lipid emulsion replaced part of the standard lipid emulsion.Liver function was tested on days 2 and 9 after surgery.Pathological examination was performed after reperfusion of the donor liver and on day 9.Clinical outcome was assessed based on the post-transplant investigations,including:(1) post-transplant mechanical ventilation;(2) total hospital stay;(3) infectious morbidities;(4) acute and chronic rejection;and(5) mortality(intensive care unit mortality,hospital mortality,28-d mortality,and survival at a one-year post-transplant surveillance period).RESULTS:On days 2 and 9 after operation,a significant decrease of alanine aminotransferase(299.16 U/L ± 189.17 U/L vs 246.16 U/L ± 175.21 U/L,P = 0.024) and prothrombin time(5.64 s ± 2.06 svs 2.54 s ± 1.15 s,P = 0.035) was seen in PUFA group compared with PN group.The pathological results showed that omega-3 fatty acid supplement improved the injury of hepatic cells.Compared with PN group,there was a significant decrease of post-transplant hospital stay in PUFA group(18.7 d ± 4.0 d vs 20.6 d ± 4.6 d,P = 0.041).Complications of infection occurred in 6 cases of PN group(2 cases of pneumonia,3 cases of intra-abdominal abscess and 1 case of urinary tract infection),and in 3 cases of PUFA group(2 cases of pneumonia and 1 case of intra-abdominal abscess).No acute or chronic rejection and hospital mortality were found in both groups.The one-year mortality in PN group was 9.1%(3/33),one died of pulmonary infection,one died of severe intra-hepatic cholangitis and hepatic dysfunction and the other died of hepatic cell carcinoma recurrence.Only one patient in PUFA group(1/33,3.1%) died of biliary complication and hepatic dysfunction during follow-up.CONCLUSION:Post-transplant parenteral nutritional support combined with omega-3 fatty acids can significantly improve the liver injury,reduce the infectious morbidities,and shorten the post-transplant hospital stay.

Atrial fibrillation in the elderly

This review discusses atrial fibrillation according to the guidelines of Brazilian Society of Cardiac Arrhythmias and the Brazilian Cardiogeriatrics Guidelines. We stress the thromboembolic burden of atrial fibrillation and discuss how to prevent it as well as the best way to conduct cases of atrial fibrillatios in the elderly, reverting the arrhythmia to sinus rhythm, or the option of heart rate control. The new methods to treat atrial fibrillation, such as radiofrequency ablation, new oral direct thrombin inhibitors and Xa factor inhibitors, as well as new antiarrhythmic drugs, are depicted.

Bacillus Licheniformis Dhs-40, a Flocculating Active Strain and Its Flocculating Activity

Microbial flocculants have become a hot spot in recent years. A bacterial strain with high flocculating activity was isolated from seawater samples. The strain was defined as Bacillus licheniformis dhs-40 by 16S rDNA identification and Biolog test. Ultrasonication test confirmed the flocculating activity of the strain was both in fermentation supernatant and cell. According to flocculating activity curve, the ideal fermentation time for collecting flocculating active substances was two days. The flocculating activity of the strain was sensitive to pH. The strain could only preserve flocculating activity while pH varied from 7 to 11. However, it could preserve flocculating activity while temperature varied from -20℃to 100 ℃ Saccharide, protein, lipid, nucleic acids qualitative test and RNase, Proteinase K treatment confirmed the flocculating active substances were proteins. Their flocculating activities were insensitive to Proteinase K.

Sulfated polysaccharides with antioxidant and anticoagulant activity from the sea cucumber Holothuria fuscogliva

Sea cucumber is a traditional nutritional food and medicinal resource with many bioactive components in China. Holothuriafuscogliva is a big sea cucumber with a rich of bioactive polysaccharides. To investigate the bioactivities of the polysaccharides from sea cucumber H. fuscogliva, we prepared the sulfated polysaccharides （HfP） from sea cucumber H. fuscogliva using a protease hydrolysis method. Antioxidant activities of HfP were investigated, including hydroxyl radical scavenging activity and superoxide radical scavenging activity. And, the anticoagulant activities of HfP were studied, including the activated partial thromboplastin time （APTT）, prothrombin time （PT） and thrombin time （TT）. The average molecular weight was 1 867.1 Da, with a sulfate content of 20.7%. In addition, the molar ratio of monosaccharide composition of HfP was Man： Rha： Glc A： Glc： Gal： Xyl： Fuc=0.083 6： 0.437： 0.134： 0： 1.182： 0.748： 1. It had a strong antioxidant activity, the hydroxyl and superoxide radical scavenging activity ECs0 of HfP was 3.74 and 0.037 mg/mL, respectively. It also showed a good anticoagulant activity in our study. The APTT of HfP was much higher than that of heparin sodium, and the PT and TT of HfP was close to that ofheparin sodium at a low concentration. Therefore, HfP shows a good antioxidant and anticoagulant activity and it may become a potential candidate of the natural antioxidant and anticoagulant and will have a good application future in health product or medicine industry.

Cyclooxygenase-2 polymorphisms and the risk of esophageal adeno-or squamous cell carcinoma

AIM： TO determine whether -1195 A→G and/or -765 G→C polymorphisms in Cyclooxygenase-2 CCOX-2） may have a risk modifying effect on the development of esophageal carcinoma in a Dutch Caucasian population. METHODS： Two study groups were recruited, 252 patients with esophageal carcinoma and 240 healthy controls, matched for race, age, gender and recruiting area. DNA was isolated from whole blood and used for genotyping. PCR products were digested with restriction enzymes and products were analyzed by agarose gel electrophoresis. Odds ratios （OR） and 95% confidence intervals （CI） were estimated. RESULTS： The distribution of the -1195A→G polymorphism was significantly different in esophageal cancer patients compared to controls. The -1195 GG genotype resulted in a higher risk of developing esophageal adenocarcinoma （OR = 3.85, 95% CI： 1.45-10.3） compared with the -1195AA genotype as a reference. The -765 G→C genotype distribution was not different between the two groups. The GG/ GG haplotype was present more often in esophageal adenocarcinoma patients than in controls （OR = 3.45, 95% CI： 1.24-9.58; with AG/AG as a reference）. The same trends were observed in patients with squamous cell carcinomas, however, the results did not reach statistical significance. CONCLUSION： Presence of the COX-2 -1195 GG genotype and of the GG/GG haplotype may result in a higher risk of developing esophageal carcinoma.

ALTERATION OF CHOLESTEROL SULFATE IN HUMAN SERA DURING THE COURSE OF PREGNANCY

Objective To determine the concentrations of cholesterol sulfate ( CS) in human sera and pla-cental villi during the course of pregnancy. And to analyze its inhibitory activity on thrombin and further characterize the functional significance of CS. Methods The concentrations of CS were determined by thin-layer chromatog-raphy (TLC) on 60 cases of normal pregnant women and 30 cases of normal placental villi. The effect of CS in human sera on the activity of thrombin was analyzed. Results The concentrations of CS in human sera gradually increased from the first to third trimester of gestation with a correlation coefficient of 0. 69, and a correlation between the concentration of CS and weeks of gestation (P <0. 01). CS was also contained in the placental villi, and its concentrations at the second and third trimester of gestations were 4. 7 and 6. 2-fold of that at the first trimester of gestation. CS inhibited the activity of thrombin. Conclusion Placental CS is one of the sources of CS in the serum , probably by shedding. From the observation that CS inhibited the activity of thrombin, the increased expression of CS may play an important role in the regulation of blood coagulation during the course of pregnancy.

Antigenicity and Hemaglutination Activity of a Recombinant Hemagglutinin-Neuraminidase of Paramyxovirus Tianjin Strain

Paramyxovirus Tianjin strain, a new genotype of Sendai virus, was isolated from the lungs of common cotton-eared marmoset that died of severe respiratory infection in the marmoset colonies. The 19.28% IgM positive rate in the young children with acute respiratory tract infection suggested a close relationship between Tianjin strain and humans. Hemagglutinin-neuraminidase (HN) is its major transmembrane glycoprotein responsible for viral attachment, penetration and release. To clear the relationship between HN structure and function of paramyxovirus Tianjin strain, rHN1, rHN2 and rHN3 overlapping the ectodomain of HN protein were expressed. Their antigenicity and hemaglutination activity, as well as cross reactivity to standard antisera against influenza virus type A, type B were analyzed. The results indicated expressed rHNs have the natural antigenicity. The segment rHN2 possesses more linear epitopes exposed on the surface of the native HN protein than found in segments rHN3 and rHN1. The hemagglutination activity of segment rHN3 is higher than that of segments rHN2 and rHN1, and partially dependent on the three-dimensional conformation of HN3 protein. Cross-reactivity between rHNs and standard antisera against influenza virus type A, type B suggested that rHNs might not be the best alternative as specific antigens to detect virus in clinical serum specimens.

Ellagic Acid-induced Hypercoagulable State in Animals:a Potentially Useful Animal Hypercoagulable Model for Evaluation of Anticoagulants

Objective To establish and evaluate a hypercoagulable animal model for the assessment of anticoagulants. Methods Forty mice, thirty-two rats, and twenty-four rabbits were randomly and equally divided into control group (saline) and three ellagic acid (EA)-treated groups (low, middle, and high doses). In the mice, bleeding time (BT) was estimated with tail transaction, and clotting time (CT) with template method. Prothrombin time (PT) and the activated partial thromboplastin time (APTT) in rats and rabbits were measured by means of Quick's one-stage assay and modified APTT assay respectively. In addition, thrombin activity was estimated in rats with PT assay using a hemagglutination analyzer. The circulating platelet aggregates were de- tected in rabbits through platelet counting and presented as the circulating platelet aggregate ratio (CPAR). Results EA shortened BT and CT in mice, PT and APTT in rats, and increased thrombin activity and CPAR, all in a dose-dependent manner. EA also brought reduction of PT and APTT in rabbits in dose- and time-dependent manners. Conclusion EA could induce hypercoagulable state through activating coagulation system and platelets in mice, rats, and rabbits.

Preparation and evaluation of enzyme encapsulated hydrogels(single gels and double network gels) and enzyme immobilized magnetic beads

In the present research,enzyme encapsulated hydrogels（single gels and double network gels）and enzyme immobilized magnetic beads,which allow high-throughput screening,were fabricated and evaluated in terms of the preservation,precision, and repeatability of enzyme activity.The fabricated gels and magnetic beads were analyzed in a 96-well microassay plate.Trypsin was successfully encapsulated in both types of gels and immobilized to the magnetic beads.However,pepsin,either encapsulated in the gels or immobilized to the magnetic beads,could not react with its substrates.The adaptability to various enzymes （e.g.,trypsin,β-glucuronidase,and CYP1A1）in the single gels and magnetic beads was superior to that in double network gels.However,the soak out of the enzymes was observed in the single gels.The double network gels could encapsulate trypsin,whereas the fabrication of the other enzymes（e.g.β-glucuronidase,CYP1A1,and pepsin）failed because of the inactivation of the enzymes by acryl amide and ammonium peroxodisulfate,which are the components of the gel formulation. The enzyme reaction in the magnetic beads exhibited the highest efficiency among the three fabrication methods.Furthermore, the stability of the enzymes immobilized to the magnetic beads was better than that fabricated by the other methods,and the activities of trypsin andβ-glucuronidase did not decline for up to one week.In addition,in the magnetic beads,the activities of trypsin andβ-glucuronidase can be well repeated.Hence,although the adaptability of the double network gels to various enzymes is currently limited,the efficiency of the enzyme encapsulation can be improved by optimizing the formulation of acryl amide gels.

Study on the anaphylactic shock induced by hemocoagulase for injection based on logistic analysis

In the present study, we aimed to explore the influencing factors of anaphylactic shock caused by hemocoagulase for injection and to provide a scientific basis for clinical safe medication. The cases reports on the adverse reactions induced by hemocoagulase for injection were collected in Chinese and foreign literatures. The clinical characteristics and influencing factors of anaphylactic shock induced by hemocoagulase for injection were evaluated by logistic regression analysis. In this study, 87 articles including 108 cases(68 cases of anaphylactic shock) were collected. Univariate logistic regression indicated that allergic constitution, daily dose, combined anesthesia, first drug delivery, post-dose time and course of treatment were positively associated with the incidence of anaphylactic shock caused by hemocoagulase for injection(P<0.05). The six above-mentioned factors were included in the multivariate logistic stepwise regression analysis to exclude the effects of confounding factors, and the results suggested that allergic constitution(P = 0.048, OR = 8.242), combined anesthesia(P = 0.024, OR = 22.675) and post-dose time(P = 0.006, OR = 20.255) were associated with the incidence of anaphylactic shock caused by hemocoagulase for injection. The clinicians should pay much more attention to risk factors that may cause anaphylactic shock, such as allergic constitution, combined anesthesia and post-dose time. The clinical pharmacists should strengthen pharmaceutical monitoring and improve the safety of medication.

In situ generated thrombin in the protein corona of zeolites： Relevance of the functional proteins to its biological impact

Adsorption of plasma proteins to nanomaterial surfaces has a great influence on their bio-functionality. However, there is limited understanding of the relationship between the functional proteins in the protein corona and the biological identity of the materials. Here we show that the in situ generated thrombin in the protein corona of a Ca-zeolite surface displays a calcium-dependent, unusually high （-3,000 NIH U/mg） procoagulant activity, which is even stable against antithrombin deactivation. Removing the encapsulated Ca^2＋ in the zeolites leads to deactivation by antithrombin. Our observations suggest that the thrombin activity can be regulated by the inorganic surface and cations. Most importantly, our discovery indicates the link between the biomolecules in the protein corona and the procoagulant activity of the materials, providing a new molecular basis for the procoagulant mechanism for zeolite hemostatics.

Comparison of fondaparinux sodium and low molecular weight heparin in the treatment of hypercoagulability secondary to traumatic infection

Purpose： To compare the effects and side-effects of fondaparinux sodium and low molecular weight heparin in patients with hypercoagulability accompanied with traumatic infection. Methods： Thirty-six patients with post-traumatic infections in our hospital intensive care center were diagnosed with hypercoagulability from February 2012 to February 2013. These patients were randomly divided into 2 groups. In group F （18 patients）, the patients were treated with fondaparinux sodium, 2.5 rag, 1/d for 11 d. In group L （18 patients）, the patients were treated with low molecular weight heparin, 4100 U, 1/12 h for 11 d. The incidence of deep vein thrombosis, bleeding events and multiple organ dysfunction syndrome （MODS） and mortality of two groups after anticoagulation therapy were analyzed. Fibrinogen, D-dimer level and activity of antithrombin Ⅲ were measured by the coagulation analyzer. Results： The incidence of deep vein thrombosis, MODS incidence and mortality were not significantly different between the two groups. The rate of bleeding evens in group F was lower than group L （p 〈 0.05）. Antithrombin Ⅲ got an upward trend after anticoagulant therapy, in which it was higher in group F than in group L on the 5th d and llth d （p 〈 0.05）. Fibrinogen levels were gradually increased, and there was no significant difference between two groups （p 〉 0.05）. D-dimer was significantly decreased after anticoagulant therapy for 5 d （p 〈 0.01 ）, and there were significant differences between two groups on the 5th d and 7th d （p 〈 0.05）. It showed no significant difference on the llth d （p 〉 0.05）. Conclusion： Fondaparinux sodium and low molecular weight heparin can effectively improve coagulopathy in patients with traumatic infection. Compared with low molecular weight heparin, fondaparinux sodium may reduce the risk of bleeding events in patients with hypercoagulability accompanied by traumatic infection.

Temperature-sensitive molecularly imprinted microgels with esterase activity

Temperature-sensitive molecularly imprinted microgels(MIGs)exhibiting esterase activity were prepared by a reverse emulsion method using dialdehyde dextran-histidine conjugate(PAD-His)as the functional macromonomer and p-nitrophenyl phosphate(NPP)as the stable transition state analogue(TSA)as well as Co2+as the coordination center.The catalytic activity of MIGs was greatly influenced by the amount of the template,and could be modulated by temperature.The hydrolysis kinetics of p-nitrophenyl acetate(NPA)in the presence of MIGs could be described by the Michaelis-Menten equation.The MichaelisMenten constant and maximum velocity were found to be 2.2×105mol/L and 2.04×10 -8mol/h,respectively.In addition,the MIGs were found to have a high catalytic selectivity to NPA.