Heparinases can produce biologically active oligosaccharides by specifically cleaving the α-(1,4) glycosidic linkages of heparin and heparan sulphate. Heparinases are divided into heparinase and heparanase. Because heparinase is an effective biocatalyst, more and more researchers pay attention to the application of heparinase in medical field in the recent years. Combined with the related research work in our group, the application value of heparinase in the medical field was summarized, such as the determination of the structure of heparin, the preparation of low-molecular-weight heparin and ultra-low-molecular-weight heparin, tumor therapy and as a heparin antagonist. In addition, we summarized the definition, source of heparinase and its application in the medicine field. Heparinases have a great application prospect in the field of medicine.
Heparin ; Heparin Lyase ; metabolism ; Heparitin Sulfate ; Oligosaccharides ; Polysaccharide-Lyases

OBJECTIVETo establish an ELISA approach to study the interaction of polysaccharides with cytokine in vitro.

METHODSThe heparin BSA complexes (HBC) were synthesized with a chemistry method and separated using a 1 X 90 cm column of Separose 4B. After identification of the complex via SDS-PAGE,the wells of ELISA plates were coated with HBC and the interaction of HBC with interferon-gamma (IFN-gamma) was detected. The effects of heparin, low molecular weight heparin (LMW heparin), chondroitin sulfate (CS), hyaluronic acid (HA) and carrageenans on the binding of HBC to IFN-gamma were tested in this system.

RESULTHuman recombinant IFN-gamma bound to heparin in a concentration dependent manner, the binding of IFN-gamma to HBC was detected at the concentration of 0.25 ng, and saturated at around 2 ng. Free heparin, LMW heparin, CS,HA and carrageenans competed for the binding of IFN-gamma to HBC with significant different ability. The IC(50)concentrations of heparin and LMW heparin were 2.40 microg/ml and 18.60 microg/ml respectively.

CONCLUSIONIFN-gamma is a cytokine with high binding affinity to heparin and carrageenans family but poor to CS-A and CS-C. ELISA is a simple, sensitive approach to detect the interaction of polysaccharides with cytokine in vitro.

Enzyme-Linked Immunosorbent Assay ; methods ; Heparin ; metabolism ; Interferon-gamma ; metabolism ; Polysaccharides ; metabolism ; Serum Albumin, Bovine ; metabolism

K5 polysaccharide of high molecular weight (HLW) can be splitted into low molecular weight (LMW) K5 polysaccharide by K5 lyase which can be used as the substrate of partial synthesis low molecular heparin sulfate (HS). To prepare recombinant K5 lyase (Elma) and analyze its biological activity. The gene of Elma was cloned by PCR amplification and was ligated with pET-28a. Then the recombinant expression vector pET-28a-Elma was transformed into Escherichia coli BL21 (DE3). After induction with 0.2 mmol/L IPTG at 16 degrees C for 5 h, Elma was successfully expressed, SDS-PAGE analysis demonstrated that the enzyme constituted more than 30% of the total cell proteins. After Ni(2+)-NTA affinity and G-75 chromatography, the purity of enzyme was more than 95%. Enzymatic activity was determined according to the change of absorbance at 232 nm per ml of the sample. The reduction of the polysaccharide molecular weight could be detected by PAGE electrophoresis. Elma can partially split HA and HS. Its optimal reatcion temperature is 37 degrees C and the optimal reaction pH is 7.0.
Bacterial Capsules ; metabolism ; Escherichia coli ; enzymology ; genetics ; Genetic Vectors ; genetics ; Heparin ; metabolism ; Lyases ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics

Animals ; Heparin ; pharmacology ; Hepatocytes ; cytology ; metabolism ; Mice ; Promoter Regions, Genetic ; genetics ; Transforming Growth Factor beta ; genetics

OBJECTIVES: The effects of ethanol and phenobarbital,which are known to affect metabolism of xenobiotics, on the formation of benzidine-and its metabolites-plasma protein adducts in rats administered benzidine were evaluated. METHODS: The experimental rats were divided into the control,ethanol and phenobar-bital groups. The experimental groups (ethanol and phenobarbital group)were pretreated with ethanol (1g/kg)or phenobarbital (80mg/kg)24 hours prior to the oral administration of benzidine (0.5mmol/kg). Blood samples were obtained from the vena cava from 5 rats in each group; and at 30 min,3 h,6 h,9 h,12 h,24 h,48 h,72 h,96 h,and 144 h after the administration of benzidine using heparin treated syringes.The plasma protein levels were separated immediately after taking blood samples. The adducts were underwent basic hydrolysis to convert them into aromatic amines. The hydrolyzed benzidine, monoacetylbenzidine, and 4-aminobiphenyl were analyzed by reverse-phased liquid chro-matography with an electrochemical detector. The quantitative amount of the metabolites was expressed by the plasma protein binding index(PBI). RESULTS: Similar to the hemoglobin adducts,the levels of the plasma protein adducts of the ethanol and phenobarbital groups (benzidine-, monoacetylbenzidine-, and 4-amino-biphenyl-PBI)were higher than those of the control group. These results are attributable to the fact that ethanol and phenobarbital induced to the plasma protein adduct formation. The N-acetylation ratio in the control group was highest at 72 h with 2.34.In the ethanol group,it was highest at 72 h with a ratio of 2.46 and was highest in the phenobarbital group at 72 h with a ratio of 2.43. The N-acetylation ratio of the plasma protein adducts was relatively lower than that of the hemoglobin adducts.The level of the plasma protein adduct increased more rapidly than the hemoglobin adducts in all experimental groups regardless of the pretreatment,and decreased rapidly after reaching the maximum level. CONCLUSION: The above results indicate that ethanol and phenobarbital increased the level of plasma protein adduct formation. The plasma protein adducts tended to decrease more rapidly than the hemoglobin adducts in the body after benzidine exposure. This results in this study result suggests that the effects of ethanol or phenobarbital need to be considered in the biochemical monitoring,and that the level of the plasma protein adducts be a more proper biomarker than the hemoglobin adducts for assessing the short term exposure to a benzidine and benzidine based dye.
Administration, Oral ; Amines ; Animals ; Environmental Monitoring* ; Ethanol* ; Heparin ; Hydrolysis ; Metabolism ; Phenobarbital* ; Plasma* ; Protein Binding ; Rats* ; Xenobiotics

OBJECTIVE: To explore the relationship between activity of plasma heparin cofactor II (HC II) and the incidence of in-stent restenosis aft er the intervention of arteriosclerosis obliterans in lower extremity. METHODS: A total of 62 patients with arteriosclerosis obliterans in lower extremity underwent femoropopliteal stent implantation. They were divided into 2 groups: A high HC II activity group (≥100%, n=40) and a low HC II activity group (<100%, n=22). All patients filled in follow up tables and conducted body examination. Possible risk factors resulting in restenosis were collected. Patients were followed up for 6 months after femoropopliteal stent implantation. RESULTS: Baseline clinical characteristics were not significantly different between the 2 groups. The degree and incidence of angiographic restenosis at the end of the 6th month after the implantation in the high HC II activity group were all significantly lower than those in the low HC II activity group (P<0.05). Multivariate analysis demonstrated that high plasma HC II activity was an independent factor in reducing the incidence of angiographic restenosis (OR=0.982, P=0.048, 95%CI, 0.966, 0.998). CONCLUSION High plasma HC II activity is an independent factor in reducing the degree of in-stent restenosis. The lower the plasma HC II activity, the severer the degree of in-stent restenosis.
Arteriosclerosis Obliterans ; surgery ; Constriction, Pathologic ; Heparin Cofactor II ; metabolism ; Humans ; Incidence ; Lower Extremity ; Risk Factors ; Stents

A 78-year-old man presented with an eight-hour history of chest distress. Electrocardiograph and serum cardiac enzymes were suggestive of acute inferior myocardial infarction with right ventricular infarction. The patient, who underwent emergency percutaneous coronary intervention, suffered from thrombocytopenia presenting with cerebral infarction and myocadial reinfarction during haparin exposure. The laboratory test for heparin-induced thrombocytopenia (HIT) specific antibodies (heparin-platelet factor, PF4) was positive. The case was diagnosed as arteries thrombosis due to heparin-induced thrombocytopenia; the patient died after cessation of heparin.
Aged ; Coronary Thrombosis ; diagnosis ; etiology ; metabolism ; Heparin ; adverse effects ; Humans ; Male ; Platelet Factor 4 ; metabolism ; Thrombocytopenia ; chemically induced ; complications

Mosquitoes secrete saliva that contains biological substances, including anticoagulants that counteract a host's hemostatic response and prevent blood clotting during blood feeding. This study aimed to detect heparin, an anticoagulant in Aedes togoi using an immunohistochemical detection method, in the salivary canal, salivary gland, and midgut of male and female mosquitoes. Comparisons showed that female mosquitoes contained higher concentrations of heparin than male mosquitoes. On average, the level of heparin was higher in blood-fed female mosquitoes than in non-blood-fed female mosquitoes. Heparin concentrations were higher in the midgut than in the salivary gland. This indicates presence of heparin in tissues of A. togoi.
Aedes/*metabolism ; Animals ; Anticoagulants/*isolation & purification ; Blood Coagulation/physiology ; Female ; Gastrointestinal Tract/*metabolism ; Heparin/*isolation & purification ; Male ; Salivary Ducts/metabolism ; Salivary Glands/*metabolism

OBJECTIVE: To investigate the expression of HPA, CK2beta and HIF-1alpha gene in nasopharyngeal carcinoma (NPC) tissues, and the correlation between their expression with the clinical characteristics of NPC and the relativity of HPA, CK2beta and HIF-1alpha gene in NPC tissues. METHOD: HPA, CK2beta and HIF-1alpha were detected with Super-Vision immunohistochemical method using antibody in 49 NPC specimens and 30 specimens with chronic nasopharyngitis tissue (CNT). RESULT: The expression of HPA, CK2beta and HIF-1alpha in NPC tissue were significantly higher than those in CNT tissue (P<0.05, separately). The expression of HPA, CK2beta and HIF-1alpha were significantly related to the TNM stage and whether recurrence or metastasis occur after treatment (P<0.05, separate ly), but there was no obvious correlation between its expression and the sex of NPC patient (P>0.05). The expression of HIF-1alpha was significantly related to the age of NPC patient (P<0.05), while HPA, CK2beta were not. The expression of HPA, CK2beta and HIF-1alpha in NPC tissue was positively correlated with each other (P<0.05, separately). CONCLUSION HPA, CK2beta and HIF-1alpha play synergetic role in development of NPC, which plays an important role in invasiveness,recurrence and metastasis of NPC. There could be a positive cooperation among HPA, CK2beta and HIF-1alpha in the carcinogenesis and development of NPC.
Carcinoma ; Casein Kinase II ; metabolism ; Female ; Heparin Lyase ; metabolism ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Male ; Middle Aged ; Nasopharyngeal Carcinoma ; Nasopharyngeal Neoplasms ; metabolism ; pathology ; Neoplasm Staging

BACKGROUND: The purpose of this prospective study is to define the effect of cardiopulmonary bypass (CPB) on the concentration of thyroid hormones and metabolites. METHODS: Blood samples were obtained from 15 patients undergoing open heart surgery at 1) pre-induction, 2) after heparinization, 3) during CPB, 4) 2 hours after CPB, 5) 24 hours after CPB and 6) 48 hours after CPB. Thyroid stimulating hormone, albumin, thyroxine (T4), free thyroxine (FT4), triiodothyronine (T3), free triiodothyronine (FT3) and reverse T3 (T3) were measured. RESULTS: Concentration of T3 significantly decreased after infusion of heparin and maintained at the decreased level until postbypass 24 hours. Concentration of FT3 significantly increased after heparin administration but maintained at a control level during CPB and decreased after postbypass 24 , 48 hours (p<0.05). Reverse T3 increased at 24 and 48 hours after CPB (p<0.05). Thyroxine decreased during CPB and return to control level after CPB. Free thyroxine did not change significantly. Thyroid stimulating hormone was significantly depressed at 24 hours after CPB (p<0.05). CONCLUSIONS: This result suggest that the thyroid function is depressed until 48 hours after CPB and it seems to be associated with abnormal metabolism of thyroid hormones.
Cardiopulmonary Bypass ; Heart* ; Heparin ; Humans ; Metabolism ; Prospective Studies ; Thoracic Surgery* ; Thyroid Gland* ; Thyroid Hormones ; Thyrotropin ; Thyroxine ; Triiodothyronine