Translocation of annexin B1 in response to the stimulation of PMA and ionomycin in cervical cancer cells.

Annexin B1 is a novel member of the annexin superfamily which was isolated from a Cysticercus cellulosae cDNA library. To investigate the physiological roles of annexin B1, we firstly performed immunohistochemical analysis on frozen Cysticercus cellulosae sections and found that annexin B1 was present not only in the tegument of the bladder wall, but also in the host-derived inflammatory layer; In addition, ELISA analysis revealed that annexin B1 could be detected in the cystic fluid of Cysticercus cellulosae and the sera of pigs with cysticercosis. These findings indicated that annexin B1 might be a secretary protein. We further constructed a pEGFP-annexin B1 plasmid and transfected it into SiHa cells. We found that GFP-annexin B1 was stimulated to translocate to the plasma membrane by phorbol 12-myristate 13-acetate (PMA). By contrast, it was induced to distribute at the plasma and nuclear membranes by treatment with calcium ionophore ionomycin. PMA increased annexin B1 membrane binding, which might facilitate exocytosis. Moreover, translocation of the protein to the plasma and nuclear membranes after stimulated by ionomycin, was predicted to be related to an additional function.

A novel, cheap and effective fusion expression system for the production of recombinant proteins.

To develop faster, less expensive methods for expression and purification of proteins, the annexin B1-intein fusion expression system was constructed. The interest proteins fused to the annexin B1-intein tag were purified in a single-step method based on the Ca(2+)-binding activity of annexin B1, and the annexin B1-intein fusion tag was removed based on the self-cleaving activity of the intein. Moreover, we found that in some cases, fusion to annexin B1 can promote the solubility of heterologous proteins. The production of soluble and highly active of interleukin-2 and low-molecular single-chain urokinase in our results proved that the system was a novel, cheap and effective fusion expression system for the production of valuable soluble recombinant proteins in Escherichia coli.

Annexin B1 at the host-parasite interface of the Taenia solium cysticercus: Secreted and associated with inflammatory reaction.

Annexin B1 is a novel member of annexin family firstly cloned by immunological screening a Taenia solium cysticercus library. To investigate the histological distribution and physiological role(s) of this protein, we first prepared a specific monoclonal antibody against annexin B1. Western blot analysis indicated that annexin B1 could be detected in cystic fluid of T. solium cysticercus and sera of pigs/humans with cysticercosis. Thus, annexin B1 might belong to the secreted members of annexins. Immunohistochemical analysis revealed that annexin B1 was mainly present in the tegument of bladders, but not in the scolex and neck; it was also detected in the surrounding host-derived layer with granulomatous infiltration. Together with previous, the presented data suggested that the protein inhibited mammalian PLA2 in vitro, and might down regulate host inflammatory responses.

Non-fusion expression in Escherichia coli: Single-step purification of recombinant human annexin A5 for detection of apoptosis.

Recombinant human annexin A5 (rh-annexin A5) was originally used to detect early stages of apoptosis in vitro. With the development of radioactive labeling and imaging techniques, annexin A5 labeled with radioactive markers can play a more important role in monitoring apoptotic cells in vivo. To obtain highly pure rh-annexin A5 with an easy and inexpensive purification approach, we constructed a pJLA503-annexin A5 expression plasmid, which could overexpress human annexin A5 in a soluble form in Escherichia coli. Then a novel purification method based on Ca2+-dependent phosphatidylserine (PS)-binding activity was established, whereby the purity of rh-annexin A5 was increased to 98%. To confirm the PS affinity of rh-annexin A5 produced by this purification protocol, a simple and reliable lipid membrane model was prepared and used in the binding test. As a probe to detect apoptosis, the fluorescein isothiocyanate-labeled rh-annexin A5 was incubated with apoptotic cells. The results showed that the labeled rh-annexin A5 possessed high affinity for PS molecule and was able to indicate different apoptotic states.

Tissue distribution of a plasmid DNA containing epitopes of foot-and-mouth disease virus in mice.

It is known that only the minority of plasmid DNAs effect a cure or prevention after intramuscular injection into host. But what is the fate of the majority? And indeed how many of the injected DNAs work? Till now, little is known about it. To answer these questions, two methods including PCR and autoradiography were used in distribution study in mice that had received a single muscular inoculation of plasmid DNA containing antigenic epitopes of foot-and-mouth disease virus. The results showed that the plasmid DNAs were distributed by blood circulation and degraded soon. The degradation ratio of super coiled plasmid DNA was 20.9% in 10 min, 34.1% in 1h, 86.8% in 1 day and 97.8% in 1 week in sera in vivo. And over a half of the whole were output in urine and faeces. The rest resided most in muscles as 'antigen pool', next in immune organs, kidney, liver, heart, lung and little in brain or gonad. About 40% or 0.5% of total plasmid DNAs, inferring to be effective, resided in muscles or immune organs, respectively. Collective results suggested that 'nude' DNA, as water injection, was characterized as quick absorbent, extensive distribution, but low utilization rate. Finally, the immune mechanism for the DNA vaccine was discussed.

Construction, expression, and characterization of a recombinant annexin B1-low molecular weight urokinase chimera in Escherichia coli.

To produce a thrombi-targeting plasminogen activator, low molecular weight single-chain urokinase gene (scuPA32k) was spliced with the full-length cDNA of annexin B1 gene (anxB1) by overlap extension method. The fused gene anxB1scuPA was ligated into pET28a vector, transformed into E. coli BL21-RIL, and then induced to express under the control of T7 promoter. The AnxB1ScuPA protein expressed amounted to 22% of the total bacterial proteins. The product was refolded, and then purified by using DEAE Sepharose fast flow ion-exchange column and Superdex S-200 gel-filtration column. HPLC analysis revealed that the final purity is about 95%. The specific activity of AnxB1ScuPA, measured as amidolytic activity, reached 100,000 IU/mg. It had a similar S2444 catalytic efficiency (kcat/Km) to ScuPA32k, and also showed high activated-platelet membrane-binding activity and anticoagulant activity, indicating that the chimera fully retained the components of enzymatic and membrane-binding activities of the parent molecules. In vivo test revealed that, the dogs administered with AnxB1ScuPA had less reperfusion time, higher reperfusion ratio, and less bleeding effects than those with urokinase. These findings indicated that AnxB1ScuPA might have advantages over current available thrombolytic agents.