ABSTRACT
Heparinase III is an enzyme that specifically cleaves certain sequences of heparan sulfate. Previous reports showed that this enzyme expressed in Escherichia coli was highly prone to aggregation in inclusion bodies and lacks detectable biological activity. In this paper, we fused a glutathione-S-transferase (GST) tag to the N-terminus of heparinase III gene and expressed the fusion protein in Escherichia coli to develop an expression system of soluble heparinase III. As a result, approximately 80% of the fusion protein was soluble. The protein was then purified to near homogeneity via one-step affinity chromatography. A 199.4-fold purification was achieved and the purified enzyme had a specific activity of 101.7 IU/mg protein. This represented 32.3% recovery of the total activity of recombinant GST-heparinase III. The maximum enzyme production was achieved when bacteria were induced with 0.5 mmol/L isopropyl-beta-D-thiogalactoside at 15 degrees C for 12 h. The enzyme showed maximum activity at 30 degrees C and pH 7.5. And the enzyme activity was stimulated by 1 mmol/L Ca2+ and 150 mmol/L NaCl.
Subject(s)
Escherichia coli , Genetics , Metabolism , Flavobacterium , Genetics , Glutathione Transferase , Genetics , Heparin Lyase , Genetics , Recombinant Fusion Proteins , GeneticsABSTRACT
Aim To investigate the effect of ultra low molecular weight heparin(ULMWH)to protect primarily cultured rat cortical neurons from the deleterious effects of the neurotoxicant glutamate (Glu)and explore the related mechanism.Methods Cortical neurons of fetal rats were cultured carefully in vitro and treated with 100 ?mol?L-1 Glu so that the protective effects of ULMWH were observed thoroughly. And then the viability of cortical neurons, morphological change together with the number of apoptotic neurons,and the concentration of intracellular free Ca2+([Ca2+]i)were measured by MTT assay, Hoechst33258 staining and Fura-2/AM double wavelength fluoremetry, respectively.Results Brief exposure of cultures to 100 ?mol?L-1Glu led to extensive neuronal death and rapid increase of [Ca2+]i.Pretreatment with ULMWH over the concentration range of 0.01~1 mg?L-1 significantly inhibited the Glu-induced neuronal cell death assessed by a MTT assay and the number of apoptotic nuclei, evidenced by Hoechst 33258 staining.Glu-induced elevation of[Ca2+]i was decreased by pretreatment with 1 mg?L-1 ULMWH, which was indicated by Fura-2/AM not only in assay medium containing Ca2+ but also in Ca2+-free assay medium.Conclusions Those results suggest that ULMWH protects the cultured neurons from Glu-induced neurotoxicity, which may be attributed to its alleviating intracellular free Ca2+ overload via suppressing intracellular Ca2+ release from internal stores induced by Glu.
ABSTRACT
w-3 polyunsaturated fatty acids, particularly docosahexaenoic acid (DHA) , are essential components for the development of human brain. A fish oil product enriched with DHA (51. 65%) was used to conduct a preliminary memory study with rats employing the active avoidance test. Three doses of DHA, 1. 0 g/kg, 200 mg/kg and 40 mg/kg, were administered orally to the rats twice daily for 20 days. The results showed that there was a positive correlation between the effects on memory ability and DHA adiministration, and the effects were dose responsive. The results also showed that DHA, at 200 mg/kg, could antagonize dysmnesia caused by scopolamine and had hypermnesia effect.
ABSTRACT
AIM To study effects of desulfated and poly sulfated heparin derivatives on rat mast cell (MC) degranulation. METHODS Different methods were used to prepare different sulfated heparin derivatives: 2 O desulfated heparin (2DeSH),N desulfated reacetylated heparin (NDeSAcH),6 O desulfated heparin (6DeSH),poly sulfated heparin (PSH). Passive MC degranulation induced by ovalbumin in rats to was employed observe the effects of different sulfated derivatives on rats MC degranulation. RESULTS All the sample groups were found of obvious inhibition of MC degranulation( P