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Levels of γ-aminobutyric acid (GABA) and glutamic acid decarboxylase 1 (GAD1), the enzyme that synthesizes GABA, are significantly increased in neoplastic tissues. However, the mechanism underlying this increase remains elusive. Instead of silencing gene transcription, we showed that the GAD1 promoter was hypermethylated in both colon and liver cancer cells, leading to the production of high levels of GAD1. GAD1 is a target gene that is silenced by H3K27me3. The key locus responsible for GAD1 reactivation was mapped to a DNA methylation-sensitive CTCF-binding site (CTCF-BS3) within the third intron of GAD1. Chromosome configuration capture (3C) analysis indicated that an intrachromosomal loop was formed by CTCF self-dimerisation in normal cells (CTCF binds to both unmethylated CTCF-BS3 and CTCF-BS2). The CTCF dimer then interacted with suppressor of zeste 12 homologue (SUZ12), which is a domain of Polycomb repressive complex 2 (PRC2), promoting the methylation of H3K27 and the silencing of GAD1 expression. This silencing was shown to be inhibited by DNA methylation in cancer cells. These findings strongly suggest that GAD1 is reactivated by DNA methylation, which provided a model for DNA methylation and the active orchestration of oncogenic gene expression by CTCF in cancer cells.

In our study, transcriptome microarrays are used to identify differentially expressed miRNAs and mRNAs in cervical cancer specimens. We find that microRNA-145 (miR-145) expression is significantly decreased in cervical cancer tissues and cell lines, and is associated with advanced cancer stages, large tumor size and moderate/poor differentiation. We show that miR-145 targets the DNA damage repair-associated gene Helicase-like transcription factor (HLTF), which is involved in radio-resistance. Moreover, miR-145 over-expression in cervical cancer cells enhances radiosensitivity in vitro and in vivo. These results indicate that targeting miR-145 may be a novel radiosensitizing strategy for cervical cancer.

Kidney stone disease is a major cause of chronic renal insufficiency. The role of long non-coding RNAs (lncRNAs) in calcium oxalate-induced kidney damage is unclear. Therefore, we aimed to explore the roles of lncRNAs in glyoxylate-exposed and healthy mouse kidneys using microarray technology and bioinformatics analyses. A total 376 mouse lncRNAs were differentially expressed between the two groups. Using BLAST, 15 lncRNA homologs, including AU015836 and CHCHD4P4, were identified in mice and humans. The AU015836 expression in mice exposed to glyoxylate and the CHCHD4P4 expression in human proximal tubular epithelial (HK-2) cells exposed to calcium oxalate monohydrate were analyzed, and both lncRNAs were found to be upregulated in response to calcium oxalate. To further evaluate the effects of CHCHD4P4 on the cell behavior, we constructed stable CHCHD4P4-overexpressing and CHCHD4P4-knockdown HK-2 cells. The results showed that CHCHD4P4 inhibited cell proliferation and promoted the epithelial-mesenchymal transition in kidney damage and fibrosis caused by calcium oxalate crystallization and deposition. The silencing of CHCHD4P4 reduced the kidney damage and fibrosis and may thus be a potential molecular target for the treatment of kidney stones.

Current literature provided information that alteration in microRNA expression impacted sensitivity or resistance of certain tumor types to anticancer treatment, including the possible intracellular pathways. The microRNA-23a (miR-23a)-regulated apoptosis in response to the 5-fluorouracil (5-FU)-induced mitochondria-mediated apoptotic pathway was determined in this study. The miR-23a expression in 5-FU-treated and untreated colon cancer cells and tissues was assessed using real-time PCR analysis. To determine the function of miR-23a in the regulation of 5-FU-induced apoptosis, cell-proliferation, cytotoxicity, and apoptosis analyses were performed. Dual luciferase reporter assay was used to identify the apoptosis-related target gene for miR-23a. The activity of caspases-3, -7, and -9 were also assessed in miR-23a antisense and 5-FU treated tumor cells. A xenograft tumor model was established to evaluate the biological relevance of altered miR-23a expression to the 5-FU-based chemotherapy in vivo. We found that the expression of miR-23a was increased and the level of apoptosis-activating factor-1 (APAF-1) was decreased in 5-FU-treated colon cancer cells compared to untreated cells. The activation of the caspases-3 and 7 was increased in miR-23a antisense and 5-FU-treated colon cancer cells compared to negative control. APAF-1, as a target gene of miR-23a, was identified and miR-23a antisense-induced increase in the activation of caspase-9 was observed. The overexpression of miR-23a antisense up-regulated the 5-FU induced apoptosis in colon cancer cells. However, the miR-23a knockdown did not increase the antitumor effect of 5-FU in xenograft model of colon cancer. This study shows that miR-23a antisense enhanced 5-FU-induced apoptosis in colorectal cancer cells through the APAF-1/caspase-9 apoptotic pathway.

Normal implantation and placental development depend on the appropriate differentiation and invasion of trophoblast cells. Inadequate trophoblast cell invasion results in pregnancy-related disorders, which endanger both mother and fetus; however, the mechanism of early placental development has not been fully explained. In this study we conducted gene expression profile analysis using mouse placental tissues at different developmental stages (embryonic day (E)7.5, E14.5 and E19.5) using series tests of cluster (STC) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analyses. Focal adhesion kinase (FAK) signalling pathway-related gene expression levels were verified using quantitative reverse transcription polymerase chain reaction and western blot. The results showed that caveolin-1 (Cav1) was downregulated in the placenta of unexplained spontaneous abortion subjects compared with that of induced abortion. Furthermore, by modulating CAV1 expression levels, CAV1 was shown to promote human trophoblast cell proliferation, migration and invasion by activating the FAK signalling pathway. These results indicate that CAV1 and the FAK signalling pathway are crucial for early placental development, which sheds new light on our understanding of the mechanisms of human trophoblast cell invasion and early development of the placenta.

Background H19 was one of the earliest identified, and is the most studied, long noncoding RNAs. It is presumed that H19 is essential for regulating development and disease conditions, and it is associated with carcinogenesis for many types. However the biological function and regulatory mechanism of this conserved RNA, particularly with respect to its effect on transcription, remain largely unknown. Methods We performed RNA pulldown, RNA immunoprecipitation and deletion mapping to identify the proteins that are associated with H19. In addition, we employed EU (5-ethynyl uridine) incorporation, immunoprecipitation and Western blotting to investigate the functional aspects of H19. Results Our research further verifies that H19 is bound to hnRNP U, and this interaction is located within the 5′ 882 nt region of H19. Moreover, H19 disrupts the interaction between hnRNP U and actin, which inhibits phosphorylation at Ser5 of the RNA polymerase II (Pol II) C-terminal domain (CTD), consequently preventing RNA Pol II-mediated transcription. We also showed that hnRNP U is essential for H19-mediated transcription repression. Conclusions In this study, we demonstrate that H19 inhibits RNA Pol II-mediated transcription by disrupting the hnRNP U–actin complex. General significance These data suggest that H19 regulates general transcription and exerts wide-ranging effects in organisms.

Alternative splicing plays critical roles in many pathophysiological processes and splicing dysregulation is a hallmark of cancer. The different isoforms may have significantly different effects on cancers. POLDIP3 is a target of ribosomal protein S6 kinase 1, and regulates DNA replication and mRNA translation. In this study, we measured the expression of an alternative POLDIP3 transcript (POLDIP3-β), which lacks exon 3 and 29 amine acids, in clinical hepatocellular carcinoma (HCC) tissues. The roles of POLDIP3-β on HCC cell proliferation, apoptosis, and migration were assessed by Glo cell viability assays, Ethynyl deoxyuridine incorporation assays, colony formation assays, TUNEL assays, Annexin V-propidium iodide staining and flow cytometry, transwell assays, wound healing assays, and in vivo xenograft growth. Our results showed that POLDIP3-β was significantly upregulated in HCC tissues compared with paired adjacent noncancerous hepatic tissues. In vitro and in vivo functional experiments results demonstrated that overexpression of POLDIP3-β drastically increased HCC cell proliferation, inhibited HCC cell apoptosis, enhanced HCC cell migration, and promoted xenograft growth. While the effects of normal POLDIP3, which contains exon 3, were much weaker. In conclusion, our study demonstrated that an alternative transcript of POLDIP3 is upregulated and functions as a critical oncogene in HCC. Selectively targeting this isoform of POLDIP3 would be a promising therapeutic strategy for HCC.

Although the use of sorafenib appears to increase the survival rate of renal cell carcinoma (RCC) patients, there is also a proportion of patients who exhibit a poor primary response to sorafenib therapy. It is therefore critical to elucidate the mechanisms underlying sorafenib resistance and find representative biomarkers for sorafenib treatment in RCC patients. Herein, we identified a long non-coding RNA referred to as lncRNA-SRLR (sorafenib resistance-associated lncRNA in RCC) that is upregulated in intrinsically sorafenib-resistant RCCs. lncRNA-SRLR knockdown sensitized nonresponsive RCC cells to sorafenib treatment, whereas the overexpression of lncRNA-SRLR conferred sorafenib resistance to responsive RCC cells. Mechanistically, lncRNA-SRLR directly binds to NF-κB and promotes IL-6 transcription, leading to the activation of STAT3 and the development of sorafenib tolerance. A STAT3 inhibitor and IL-6-receptor antagonist both restored the response to sorafenib treatment. Moreover, a clinical investigation demonstrated that high levels of lncRNA-SRLR correlated with poor responses to sorafenib therapy in RCC patients. Collectively, lncRNA-SRLR may serve as not only a predictive biomarker for inherent sorafenib resistance but also as a therapeutic target to enhance responses to sorafenib in RCC patients.

Affibodies are a group of affinity proteins that are based on a 58-amino-acid residue protein domain derived from one of the IgG-binding domains of staphylococcal protein A. A single human IgA affibody with high IgA affinity has been generated by directed evolution. It remains interesting whether tandem IgA affibody proteins could increase binding capacity. Here, we report the generation of multiple tandem IgA affibodies by directed evolution using a combinatorial phage library displaying the IgA affibody A1 and/or A2 linked with three random amino acids. These affibodies exhibited markedly increased IgA binding capacity, as shown by enzyme linked immunosorbent assay, immunoblotting and surface plasmon resonance assays. We further showed that these tandem IgA affibodies displayed preferential binding to intact IgA molecules compared to individual IgA chain, suggesting intramolecular binding avidity. Our data demonstrates that artificial multiple tandem human IgA affibodies with relevant biological binding avidity were successfully yielded by phage-based molecular evolution. These results have broad implications for the design and development of binding proteins that target important biological molecules.

Pre-eclampsia is a leading cause of maternal and foetal morbidity and mortality worldwide. Insufficient uteroplacental oxygenation is believed to be responsible for the disease. However, what molecular events involve in hypoxic responses and how they affect placental development remain unclear. Recently, miRNAs have emerged as a new class of molecules in response to hypoxia. We show here that the expression of microRNA-210 (mir-210) is up-regulated in patients with pre-eclampsia, as well as in trophoblast cells cultured under hypoxic conditions. Ectopic expression of mir-210 inhibited the migration and invasion capability of trophoblast cells. Ephrin-A3 and Homeobox-A9, which related with cell migration and vascular remodelling, were then experimentally validated as the functional targets of mir-210 both in vivo and in vitro. Using luciferase reporter, chromatin immunoprecipitation (ChIP) and small interfering RNA (siRNA) experiments, we finally identified a new transcriptional mechanism that the overexpression of mir-210 under hypoxia was regulated by NF-κB transcriptional factor p50, apart from the well-known HIF 1α. Taken together, our study implicates an important role for mir-210 in the molecular mechanism of pre-eclampsia.

DNA vaccines are widely used against infectious agents for their ability to induce both humoral and cellular immune responses. However, safety concerns regarding autoimmune responses to DNA vaccines, particularly to certain plasmids, should not be neglected. In this study, we serendipitously found that mice inoculated with pcDNA3-ANXB1 (pcDNA3-b1) developed autoimmunity, which did not happen in pVAX-ANXB1 (pVAX-b1) inoculated mice. We also employed proteomics approaches to investigate the distinction between the two groups of DNA vaccine immunized mice. Five different proteins with three-fold or greater changes were separated and identified by two-dimensional electrophoresis. Our study verified the safety of the DNA vaccine and unveiled the underlying potential molecular mechanism of DNA vaccine delivery.

Annexin B1 is a novel Ca(2+)-dependent phospholipid-binding protein from metacestodes of Taenia solium and has been shown to have many potential biomedical applications. Although annexin B1 has been produced successfully in Escherichia coli, the purified protein has poor stability at room temperature, which has hindered our attempts to further study its structure-function relationship. To increase the stability of the protein, the construction and purification procedures were examined and changed to hopefully increase its effectiveness. In this study, we describe a new recombinant annexin B1 expressed with a hexahistidine tag fused to its N-terminal end, which was purified to homogeneity in two steps using immobilized metal affinity followed by size exclusion chromatography. The final yield was approximately 23 mg/L of bacterial culture. Isoelectric focusing and mass spectrometry analysis showed that the protein purified by this method was quite stable at room temperature, even greater than 3 days later. A series of functional tests indicated that the recombinant protein had high anticoagulant activity, and fluorescence-labeled annexin B1 could bind to the outer membranes of apoptotic mammalian cells and efficiently detect them in the early stages of apoptosis.

Foot-and-mouth disease virus (FMDV) is the causative agent of foot-and-mouth disease, a severe, clinically acute, vesicular disease of cloven-hoofed animals. RNA interference (RNAi) is a mechanism for silencing gene expression post-transcriptionally that is being exploited as a rapid antiviral strategy. To identify efficacious small interfering RNAs (siRNAs) to inhibit the replication of FMDV, candidate siRNAs corresponding to FMDV VP1 gene were designed and synthesized in vitro using T7 RNA polymerase. In reporter assays, five siRNAs showed significant sequence-specific silencing effects on the expression of VP1-EGFP fusion protein from plasmid pVP1-EGFP-N1, which was cotransfected with siRNA into 293T cells. Furthermore, using RT-qPCR, viral titration and viability assay, we identified VP1-siRNA517, VP1-siRNA113 and VP1-siRNA519 that transiently acted as potent inhibitors of FMDV replication when BHK-21 cells were infected with FMDV. In addition, variations within multiple regions of the quasispecies of FMDV were retrospectively revealed by sequencing of FMDV genes, and a single nucleotide substitution was identified as the main factor in resistance to RNAi. Our data demonstrated that the three siRNA molecules synthesized with T7 RNA polymerase could have transient inhibitory effects on the replication of FMDV.

HBc-VLP can be used in an epitope presentation system to carry foreign epitopes and mimic live virus in order to study viral particle uptake, virion-mediated activation and antigen presentation by dendritic cells. In this study, a multifunctional mAb was produced using a novel research strategy. A truncated HBc-VLP bone vector with a special conformation was used as an immunogen and the target hybridoma cell lines were screened by a series of tests; including ELISA, Western blot, and cellular immunofluorescence based on the epitope presentation system. The screened monoclonal antibody was used to identify the HBc-VLP vector, a fusion HBc-VLP vaccine, and intracellular HBV capsids. The new strategy facilitated acquisition of the desired mAbs and will serve as a reference for other VLP-related research.

Background The effect of formaldehyde treatment on the recovery of fetal DNA from maternal plasma is controversial. We evaluated the effect of formaldehyde and investigate the underlying mechanism. Methods Blood samples from pregnant women were treated or not treated with formaldehyde, and processed at different times. Total and fetal DNA in plasma was quantified by real-time polymerase chain reaction. Death and lysis of blood cells were assayed by trypan blue exclusion test. Plasma DNase activity was determined by the radial enzyme-diffusion method. Results Formaldehyde addition showed no effect on the percentage of fetal DNA in samples processed 6 h after blood collection. In samples processed at 36 h, formaldehyde addition inhibited blood cell lysis and nuclease-mediated DNA degradation, thus markedly decreasing the concentration of total DNA and increasing the recovery of fetal DNA. The median (interquartile range) percentage of fetal DNA increased from 4.6% (3.8–6.8%) to 13.1% (10.3–17.0%). Conclusion The effect of formaldehyde on the percentage of fetal DNA in maternal plasma depends on processing time and is associated with prevention of cell lysis and inhibition of plasma DNase activity.

The biological mechanism of a recent discovered association of type 2 diabetes with the ACAA-insertion/deletion polymorphism at the 3'UTR of the IGF2R gene has remained unclear. A very recently emerging novel polymorphic control layer by microRNAs (miRNAs) makes it possible to elucidate this issue. In this study, a prediction by web tools MicroInspector and miRanda demonstrated that DNA sequence polymorphism (DSPs) ACAA-insertion/deletion in IGF2R 3'UTR is located within the hsa-miR-657 and hsa-miR-453 binding sites. And luciferase reporter assay revealed that hsa-miR-657 acts directly at the 3'UTR of the IGF2R. Furthermore, ACAA-deletion exerted a further repression compared with ACAA-insertion, indicating that hsa-miR-657 regulates IGF2R gene expression in a polymorphic control manner. Importantly, we also demonstrated that hsa-miR-657 can translationally regulate the IGF2R expression levels in Hep G2 cells. Thus, our findings testify the possibility that the ACAA-insertion/deletion polymorphism may result in the change of IGF2R expression levels at least in part by hsa-miR-657-mediated regulation, contributing to the elucidation for the pathogenesis of type 2 diabetes and raise the possibility that miRNAs or in combination with functional DNA sequence polymorphism may be valuable in the treatment of human type 2 diabetes.

Annexin B1, a novel Ca2+-dependent PS-binding protein, has been shown to have a high affinity for PS exposed on the surface of apoptotic cells. To develop and bioevaluate an annexin B1 based PS-targeting radiotracer, annexin B1 was radiolabeled with (99m)Tc using HYNIC as a bifunctional chelator. Binding assays with activated platelets and apoptotic SP2/0 cells were carried out to evaluate the in vitro biological activity of (99m)Tc-HYNIC-annexin B1. Biodistribution of this radioligand was studied in normal mice. Dexamethasone-induced murine thymus apoptosis and fas-mediated murine liver apoptosis models were used to investigate the ability of radiolabeled annexin B1 to detect apoptosis in vivo. The labeling procedure yielded a compound with up to 98% radiochemical purity and good in vitro stability. The in vitro binding assays indicated that (99m)Tc-HYNIC-annexin B1 retain its PS-binding activity. Biodistribution of the compound in mice showed that (99m)Tc-HYNIC-annexin B1 is rapidly cleared from the blood and predominantly accumulates in the kidney. The marked increase in dexamethasone-treated murine thymus uptake and fas-mediated murine liver uptake correlated with histologic evidence of apoptosis. These data suggested that (99m)Tc-HYNIC-annexin B1 retain its in vitro and in vivo biological activities. This radiotracer may therefore be useful as a novel radioligand for the noninvasive detecting of PS externalization associated with apoptosis.

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.

We previously reported cloning of theTaenia soliumannexin B1 gene from a metacestode cDNA expression library and demonstrated that it acts as a protective antigen for effective vaccine development against cysticercosis. In the present study we produced recombinant annexin B1 and antiserum against the protein to investigate its structural and functional properties. Western blotting of metacestode fractions indicated thatT. soliumannexin B1, similar to vertebrate annexins, associates with acid phospholipids in the presence of Ca2+. This property was confirmed by the recognition of apoptotic cells by labeled annexin B1. CD spectroscopy results demonstrated that α-helices are the main secondary structures of the protein. Ca2+binding increases the α-helix content and causes significant thermal stabilization with a melting temperature increase of approximately 10°C. Functional Ca2+-dependent phospholipid binding sites of annexin B1 were investigated using mutant proteins. By changing a conserved acidic amino acid residue that putatively combines Ca2+in each domain of annexin B1 singly or in combination, we found that Ca2+binding in the first domain is more important than that at the other Ca2+binding sites. Annexin B1 is a metacestode stage-specific antigen, with the protein being mainly localized in the teguments and surrounding cyst wall ofT. soliummetacestodes, suggesting a role in the parasite-host interaction.

The major aim of the project was to develop the virus-like particles (VLPs) displaying single or multi-epitope of hepatocellular carcinomas (HCC) in Escherichia coli and to evaluate the effect on inducing Ag-specific CD8(+) T cell response and antitumor efficacy as candidate vaccines. To this end, hepatitis B virus core (HBc) particles were used as a carrier of HCC epitopes. Four HCC epitopes MAGE-1(278-286aa), MAGE-3(271-279aa), AFP1 (158-166aa) or AFP2 (542-550aa) were fused to the 3' terminus of the truncated HBV core gene, respectively, or conjunctively. Not all recombinant plasmids led to expression of chimeric proteins in expression strain E. coli BL21 (DE3), but chimeric proteins which are expressed in inclusion bodies resulted in the formation of complete "mature" VLPs. E. coli-derived truncated HBc(1-144) chimeric protein self-assembled into VLPs that both morphologically and physically are similar to the wild-type ones and they still remained activity after purification and refolding from 6M urea solution. We also showed that they could be internalized and presented by DCs in vitro. Additionally, DCs pulsed with the chimeric HBc-VLPs could induce stronger CTL activity and greater IFN-gamma secretion by responding T cells compared with peptid-pulsed DCs. In the B16-pIR-HH tumor therapy model, the growth of established tumors was significantly inhibited by immunization using VLP-pulsed DCs, resulting in significantly higher survival rate of immunized animals. Thus, the results of the current study have demonstrated the principal possibility of using VLP on the basis of HBcAg for creation of a new type of HCC-specific immunogen.

The p53 promoter-based green fluorescent protein (GFP) and luciferase reporter gene assays have been established for detecting DNA damage induced by genotoxic agents. To evaluate the system, NIH3T3 cells transfected with either pHP53-GFP or pMP53-GFP construct were treated with mitomycin or 5-fluorouracil. Expression of the GFP reporter gene was significantly and specifically induced in the cells exposed to mitomycin or 5-fluorouracil. Then we treated NIH3T3 cells harboring pHP53-Luc or pMP53-Luc vector with mitomycin, 5-fluorouracil or cisplatin at various concentrations. Similarly, exposure of the cells to these agents with genotoxic potentials resulted in a dose-dependent induction in luciferase reporter gene expression. Thus, these in vitro reporter gene assays could provide an ideal system for quick assessment or screening of agents with genotoxic potential.

DNA vaccines have been widely reported to elicit both effective humoral and cellular immune responses, but the mechanisms of antigen processing and presentation in DNA immunization is still ambiguous. Aiming to molecular mechanisms involved in DNA immunization, comparative serum proteomics was introduced to discover differentially expressed proteins after different immunizations. Using two-dimensional electrophoresis and matrix-assisted laser desorption ionisation-time-of-flight mass spectrometry, 23 three-fold or greater up-regulated proteins were separated and identified, including 14 from ANXB1 DNA immunized mice and 9 from annexin B1 protein immunized mice. The histocompatibility class I molecule H2-Q10 (HA10_MOUSE) and proteasome activator PA28 alpha-subunit (PSME1_MOUSE) were found up-regulated in ANXB1 DNA immunized mice, which may contribute to the augmented activation of T lymphocytes. These proteins may serve as potential surrogate markers of successful vaccination and provide research targets for molecular mechanisms of vaccinology.

In this study, we found that H19 is the most strongly differentially expressed long noncoding RNA (lncRNA) during liver development. H19 may inhibit the proliferation of fetal liver cells by blocking the interaction between heterogeneous nuclear ribonucleoprotein (hnRNP) U and actin, which results in gene transcriptional repression. Based on ChIP-seq analysis, we found that genes involved in the Wnt signaling pathway are enriched among hnRNP U-binding genes. Further investigation demonstrated that hnRNP U has opposing effects on cell proliferation and Wnt/β-catenin signaling pathway activity compared to H19 and that hnRNP U is very important in this process.

A novel annexin gene was isolated from the Taenia solium cDNA library by degenerate PCR. The purified protein was generated by hydrolysis with thrombin in glutathione S-transferase (GST) affinity chromatography following expression in GST tagged pGEX-5T vector. It shares the common structural features of the annexin family and specially possesses two unique fragments between repeating domains II and III which do not exist in other annexin family members revealed by aligning and homology modeling, hence it was designated as Tso ANXB2 according to the new nomenclature of annexins. According to the parasitic behaviors of the origin, a series of coagulation assays was performed, indicating that Tso ANXB2 inhibits extrinsic blood coagulation pathway and platelet aggregation also has platelet binding activity. Nevertheless, the mutant protein deleting the consensus coagulation-related KGD motif of Tso ANXB2 showed significant decreasing platelet binding and anticoagulation activity.

Neuraminidase (NA) is one of the surface glycoproteins of influenza virus. The immune response induced by NA DNA in the mouse model has been proved, in our previous study, to be able to provide an adequate protection against the challenge with a homologous virus and a crossprotection against the challenge with a heterologous virus of the same subtype. In this paper, a series of NA plasmid truncates, with the nucleotides at the 5'- or 3'-terminal of A/PR/8/34 (H1N1) NA deleted serially, were constructed by PCR. BALB/c mice were immunized with the plasmid truncates and challenged with homologous virus at a lethal dosage. The essential sequence of NA DNA to provide protection against the influenza virus was explored by observing the survival rates, serum anti-NA antibody titers, and lung virus titers of the mice. The result showed that, along with the increasing number of nucleotides deleted serially at the 5'- or 3'-terminal of NA DNA, the antibody titer induced by NA DNA decreased. NA DNA lost its protection against the influenza virus when 60 nucleotides (or 20 amino acids) at the 5'-terminal or 66 nucleotides (or 22 amino acids) at the 3'-terminal were deleted. The nt58-1299 of NA DNA may play an important role in protection against influenza virus.

Aim: To investigate the effects of CpG-oligodeoxynucleotide (CpG-ODN) on the formation of macrophage foam cells and related gene expression. Methods: A gene expression profile was examined by microarray techniques, and mRNA expression was detected by reverse transcriptase polymerase chain reaction (RT-PCR). The cholesterol and cholesteryl ester contents of cells were determined by high performance liquid chromatography. Results: CD36, LPL, and Fcgamma2b, which were related to lipid metabolism and the formation of macrophage foam cells, were upregulated after CpG-ODN stimulation. The mRNA expression related to the formation of foam cells was confirmed by semiquantitative RT-PCR. Moreover, histochemical analysis confirmed that lipid deposits inside cells increased after CpG-ODN treatment. However, using flow cytometry, we found that CpG-ODN had no effect on the expression of membrane receptors. Conclusion: CpG-ODN up-regulated the expression of genes in macrophage foam cell formation.

The S2 domain of the severe acute respiratory syndrome coronavirus (SARS-CoV) spike (S) protein is responsible for fusion between virus and target cell membranes, and is expected to be immungenic. In this study, we investigated the immune responses against the S2 subunit in BALB/c mice, which were vaccinated either with plasmid DNA encoding the S2 domain (residues 681-1120), the recombinant S2 fragment (residues 681-980) in incomplete Freund's adjuvant, or with inactivated SARS-CoV. The increased number of specific cytotoxic cells (CTLs) and the high titer of specific antibody showed stimulation of both arms of the immune system in these groups. The shift in cytokines suggested that Th1-polarized immune response was induced by plasmid pCoVS2, meanwhile the Th2-dominant response was induced by recombinant S2 fragment and inactivated vaccine. However, the titer of neutralizing antibodies was only detectable in mice immunized with inactivated virus, but not with pCoVS2 plasmid. Taken together, the S2 domain could induce specific cellular immune response and a high level of total IgG but little neutralizing antibodies against infection by SARSCoV.

Annexin B1 is a novel member of the annexin family of Ca2+- and phospholipid-binding proteins from Cysticercus cellulosae. To obtain high quality annexin B1 for biochemical and biophysical analyses, its cDNA was cloned into the prokaryotic expression vector pJLA503 and the translation initiation codon was immediately under the control of the inducible bacteriophage lambda promoters P(R) and P(L). After induction by shifting temperature, large amounts of non-fusion protein were produced in Escherichia coli in a soluble form. The recombinant protein was purified to homogeneity by means of two subsequent ion-exchange chromatographic steps. The final yield was about 25 mg/L bacterial culture. Western blot analysis showed that recombinant annexin B1 was specifically recognized by serum of pigs infected with cysticercosis. Secondary structure predictions from circular dichroism spectroscopy indicated that alpha-helix is the main secondary structure of the protein. In anticoagulant assays, the recombinant non-fusion protein exhibited dose-dependent effects in modified kaolin partial thromboplastin time (KPTT) prolongation and doubled the clotting time of control human plasma at 60 microg/ml. The expression, purification, and initial characterization of annexin B1 set an important stage for further characterization of the protein.

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 ex- pressed 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 (k cat /K m ) to ScuPA32k, and also showed high activated-platelet membrane-binding activity and anticoagulant activity, indicating that the chi- mera 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 reperfu- sion ratio, and less bleeding effects than those with urokinase. These findings indicated that AnxB1ScuPA might have advantages over current available thrombolytic agents.

About 61-72% of transcribed regions possess long noncoding RNAs in antisense orientation (Antisense long noncoding RNAs, aslncRNAs). However, the function of aslncRNAs in HCC remains unclear. We found numerous aslncRNAs were deregulated and might be involved in regulatory gene-net of HCC. The PCNA-AS1, antisense to PCNA, is significantly up-regulated in HCC and could promote tumor growth in vitro and in vivo. The effects of PCNA-AS1 rely on regulation of PCNA via forming RNA hybridization to increase PCNA mRNA stability. We concluded that aslncRNAs might act as upstream regulators in HCC and PCNA-AS1 could serve as a novel therapeutic target.

In recent years, long noncoding RNAs (lncRNAs) have been investigated as a new class of regulators of biological function. A recent study reported that lncRNAs control cell proliferation in hepatocellular carcinoma (HCC). However, the role of lncRNAs in liver regeneration and the overall mechanisms remain largely unknown. To address this issue, we carried out a genome-wide lncRNA microarray analysis during liver regeneration in mice after 2/3 partial hepatectomy (PH) at various timepoints. The results revealed differential expression of a subset of lncRNAs, notably a specific differentially expressed lncRNA associated with Wnt/β-catenin signaling during liver regeneration (an lncRNA associated with liver regeneration, termed lncRNA-LALR1). The functions of lncRNA-LALR1 were assessed by silencing and overexpressing this lncRNA in vitro and in vivo. We found that lncRNA-LALR1 enhanced hepatocyte proliferation by promoting progression of the cell cycle in vitro. Furthermore, we showed that lncRNA-LALR1 accelerated mouse hepatocyte proliferation and cell cycle progression during liver regeneration in vivo. Mechanistically, we discovered that lncRNA-LALR1 facilitated cyclin D1 expression through activation of Wnt/β-catenin signaling by way of suppression of Axin1. In addition, lncRNA-LALR1 inhibited the expression of Axin1 mainly by recruiting CTCF to the AXIN1 promoter region. We also identified a human ortholog RNA of lncRNA-LALR1 (lncRNA-hLALR1) and found that it was expressed in human liver tissues. Conclusion: lncRNA-LALR1 promotes cell cycle progression and accelerates hepatocyte proliferation during liver regeneration by activating Wnt/β-catenin signaling. Pharmacological intervention targeting lncRNA-LALR1 may be therapeutically beneficial in liver failure and liver transplantation by inducing liver regeneration. (Hepatology 2013;58:739–751)

Preeclampsia is a common pregnancy complication that is associated with maternal perinatal morbidity and mortality. Because of its early onset (before 34 weeks) and the potential for serious outcomes, severe, early-onset preeclampsia (sePE) should be regarded as a different form of preeclampsia. It is an important cause of preterm birth and fetal growth restriction and adverse maternal and neonatal outcomes. As there is no diagnostic test yet available for this disease, we used a proteomic approach to identify novel plasma biomarkers for developing severe, early-onset preeclampsia. We conducted case-control studies comparing nulliparous women with severe preeclampsia requiring delivery prior to 34 weeks of gestation with healthy nulliparous women matched by gestational age at sampling. Plasma was depleted of albumin and IgG and analyzed by two-dimensional gel electrophoresis (2DE). Seven specific plasma proteins for early-onset preeclampsia were detected by mass spectrometry had statistically significant expression differences when compared to controls. The expression of complement component C4A and apolipoprotein A-I were validated by immunoblotting. The complement component C4A in the plasmas of sePE women is lower than the severe, late-onset PE (slPE) women [mean ± SD; 3.05 ± 0.14 times reference level (normal/sePE) in sePE women vs. 2.73 ± 0.10 times reference level (normal/slPE) in slPE women, P < 0.05]. Apolipoprotein A-I is higher in sePE women than slPE women [mean ± SD; 1.58 ± 0.14 times reference level (sePE/normal) in sePE women vs. 1.04 ± 0.16 times reference level (slPE/normal) in slPE women, P < 0.05]. Furthermore, C4A can accurately distinguish severe PE (sePE and slPE) from mild PE (mePE and mlPE) and was proved by the results of ELISA. Further studies have been done to determine the relation between PE and hypoxia. JAR cells were cultured under hypoxia for 72 h. Total cellular proteins were gathered and lysed. Lower C4A and higher apolipoprotein A-I had been observed in JAR of hypoxia conditions than normoxia conditions through western blotting. The result proved that PE is correlated with hypoxia. In summary, C4A and apolipoprotein A-I are able to function as markers to distinguish ePE women from lPE women, and severe PE from mild PE, or perhaps even as disease predictors that might become relevant for diagnostics.

The demonstration that naked plasmid DNA can induce strong immune responses in mice has attracted considerable attention in the vaccine community. However, similar immunizations have been less/not effective in clinical trials during the past decade, and the underlying mechanisms remain unknown. In this study, we hypothesized that some DNA-binding proteins in human serum may serve as host barriers, responsible for the low efficiency of plasmids’ transfection in vivo. Using proteomics, we showed that human serum amyloid P component (hSAP) is specifically present in human DNA–protein complexes. Further analysis indicated that hSAP effectively binds plasmid DNA, inhibits DNA transfection into somatic cells and facilitates the endocytosis of DNA by macrophages, whereas mouse SAP (mSAP) has similar, but much weaker, activities. In the presence of hSAP, the plasmid DNA expression in vivo and plasmid DNA-induced immune responses also significantly decreased. Therefore, our results suggest that hSAP contributes to extracellular DNA clearance and the inhibition of plasmid DNA transfection in vivo. This mechanism may be partly responsible for the insufficient immune responses to DNA vaccination in human beings; therefore, it may serve as a novel target for the improvement of DNA vaccines and DNA-based gene therapy.

Hepatitis B virus core protein virus-like particles (HBc-VLPs) act as a strong immunogen and are suitable for uptake by dendritic cells (DCs), in which they directly promote DC maturation and migration. To illustrate the utility of global proteomic analysis techniques in elucidating the molecular events that are altered in HBc-VLP-pulsed bone marrow-derived DCs (BMDCs) and to gain a better understanding of the molecular mechanisms of capture and processing of HBc-VLP-pulsed BMDCs, an antigen (Ag) delivery system based on HBc-VLP-pulsed BMDCs was developed. Two-dimensional electrophoresis (2-DE) and tandem mass spectrometry (MS/MS) analyses were utilized to analyze the differential protein expression patterns between HBc-VLP-pulsed and untreated BMDCs. Protein spots with significantly altered expression levels were detected, identified and validated. The results showed that exogenous HBc-VLPs were phagocytosed efficiently by BMDCs and enhanced the efficacy of BMDC maturation and Ag presentation, VLPs also induced high levels of Ag-specific CD8(+) T cells that displayed high cytotoxic T lymphocyte (CTL) activity in vivo. Several differentially expressed proteins, including growth factor receptor bound protein 2 (Grb2) and annexin A2 (AnxA2), were detected by proteomic analysis, identified by mass spectrometry and validated by western blot.

Annexin B2 (AnxB2) is a novel member of the annexin family of Ca(2+)- and phospholipid-binding proteins from Cysticercus cellulosae. To obtain highly pure AnxB2 with an easy and inexpensive purification approach, its cDNA was cloned into the prokaryotic expression vector pJLA503 and the translation initiation codon was immediately under the control of the inducible bacteriophage lambda promoters P(R) and P(L). After induction by shifting temperature, large amounts of non-fusion protein were produced in Escherichia coli in a soluble form. Then a novel purification method based on Ca(2+)-dependent phosphatidylserine (PS)-binding activity was established, whereby the purity of AnxB2 was increased to 98.7%. Western blot analysis showed that recombinant AnxB2 was specifically recognized by serum of pigs infected with cysticercosis. In vitro test showed that, the recombinant AnxB2 had anticoagulant activity and platelet binding activity. The expression, purification, and initial characterization of AnxB2 set an important stage for further characterization of the protein.

Many recent studies have indicated that virus-like particles (VLPs) have many potential applications in the fields of vaccine development and gene therapy. However, we still know little about the subtle mechanisms involved in the presentation of VLPs by antigen presenting cells (APCs). To illustrate the mechanisms, we utilized two-dimensional electrophoresis and tandem MS to compare and identify differentially expressed proteins between hepatitis B virus core antigen VLP (HBc-VLP)-pulsed and control RAW264.7 cells. Of the 25 spots identified as differentially expressed ( p < 0.05) between the two cell lines, 11 (corresponding to 11 unique proteins) were positively identified. Further analysis of two proteins, prohibitin and heat shock protein 70, confirmed that these proteins are expressed at higher levels in HBc-VLP-pulsed RAW264.7 cells compared with control cells. The proteins identified in this study will be useful in revealing the mechanisms that underlie VLP-APC interactions. Overall, this study also provides some useful suggestions for vaccine development and gene therapy.

In addition to conventional vaccination, DNA-mediated immunization has been developed as an alternative approach in the prevention and treatment of different infectious diseases, including hepatitis B. To define sets of serum protein and metabolite biomarkers that could be employed to determine the efficacy and safety of DNA vaccines, an integrated multiple systems biology approach was undertaken on mice immunized with DNA vaccine, recombinant protein, plasmid vector, and phosphate-buffered solution. Their sera were analyzed by two-dimensional electrophoresis and HPLC coupled with time-of-flight mass spectrometry. We detected an increase in phytosphingosine, dihydrosphingosine, palmitoylcarnitine, and ceramide in the sera of DNA-vaccinated mice. Several protein molecules were found to be altered in DNA-vaccinated mice, including apolipoprotein A-I precursor. Taken together, these results indicated that DNA vaccine stimulated hepatic sphingolipid synthesis, which may have altered the structure of circulating lipoproteins and promoted atherogenesis. This study also underscores the power of metabolomics and proteomics in the definition of DNA-vaccine-mediated metabolic phenotypes.

The interest in understanding interaction between the toll-like receptor and the viral infection has increased because of its importance in disease control. Here we have cloned the porcine TLR7 (pTLR7) cDNA encoding for 1050 amino acids. The pTLR7 Exhibits 90, 87, 87, 86, 84 and 78% similarity to TLR7 from cattle, dog, horse, cat, human and mouse, respectively. RT-PCR data suggested that pTLR7 mRNA was mostly synthesized in secondary lymphoid tissues (spleen, lymph node and tonsil) and antigen-presenting cells (macrophage and B cell). The cellular experiments indicated that immunostimulatory RNA oligonucleotides (ORN) found in foot-and-mouth disease virus (FMDV) activated nuclear factor-kB and induced the expression of IFN-α via pTLR7. We found that the ORN induced Th1-type cytokines and activated immunocytes in PBMC. The study provides foundation for further investigation in pTLR7 interactions with its ligands and interactions between the porcine immune system and pathogens.

Background Chitosan has been shown to possess useful properties such as non-toxicity, high biocompatibility and non-antigenicity that offer advantages for vaccine delivery systems. In this study, we prepared novel chitosan derivative nanoparticles as DNA vaccine carriers and the potential and mechanism of the DNA-nanoparticle complexes in inducing augmented immune responses were explored. Methods The pVAX(HBc)DNA-nanoparticle complexes as vaccine delivery systems were studied in several aspects: the protection against DNase I degradation was measured by an in vitro inhibition assay; the sustained expression of the plasmid in vivo was determined by RT-PCR; the elevated uptake efficiency by phagocytes was observed with confocal microscopy; the biocompatibility was evaluated by cytotoxicity and histology assay; the complexes were administrated to C57BL/6 mice and the humoral and cellular immune responses were evaluated by ELISA, IFN-γ production and cytolytic T lymphocyte (CTL)-specific lysis assay. Results The remaining relative activity of DNase I after inhibition varied from 32.3% to 77.6%. The complexes were observed with higher uptake efficiency by phagocytes than naked DNA. Three types of nanoparticles did not induce significant cytotoxicity at concentrations ⩽400 µg/ml. No specific histological alteration related to the injection of the complexes was observed. The formulations of DNA-nanoparticle complexes significantly enhanced the immunogenicity in several parameters: elevated antibody production, higher level of IFN-γ secretion, and augmented specific cell lysis. Conclusions This study demonstrated the potential of the novel chitosan derivative nanoparticles for safe and effective DNA vaccine delivery. Copyright © 2007 John Wiley & Sons, Ltd.

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.

Annexin B1 has many potential biomedical applications based on its high affinity for negatively charged phospholipid (phosphatidylserine, PS) in the presence of physiological concentrations of calcium. Low immunogenicity is prerequisite for the in vivo application of a nonhuman protein as a novel-imaging agent. In the present study, three sequence-deleted mutants with different numbers of functional domains were designed and expressed according to the predicted three-dimensional structure of annexin B1. The mutants of annexin B1, as well as the wild-type annexin B1, were expressed as Glutathione-S-transferase (GST)-fusion proteins. Two mutants with their purity above 80% could be obtained after one-step primary purification procedure on basis of the PS-binding activity. The immunogenicity of the two mutants was evaluated in mice by detecting the titers of elicited antigen-specific IgG. A member of three mutants of annexin B1, M12, which involved N-terminal amino-acid sequence and double functional domain I and II of annexin B1, was finally selected to detect apoptosis that is due to its lowest immunogenicity among the candidate mutants. Flourescein isothiocyanate-labeled M12 could bind the outer membranes of apoptotic cells and discriminate apoptotic cells in the early stage from necrotic cells when used with propidium iodide. (99m)Tc-labeled M12 could recognize the apoptotic hepatocytes induced by anti-Fas antibody treatment. Our data in vitro and in vivo demonstrated that M12 could be applied as a promising agent for the detection of apoptosis.

To obtain a thrombus-targeted plasminogen activator with high affinity for activated platelets and enhanced thrombolytic and antithrombotic potency, we engineered a sequence encoding RGDS (Arg-Gly-Asp-Ser) peptide into the loop between domains II and III of the sequence-deleted mutant of annexin B1 and then constructed a chimaeric plasminogen activator gene mAnxB1-RGDS-ScuPA by fusing ScuPA32k [low-molecular-mass single-chain urokinase (32 kDa)] with the N-terminus. The chimaeric protein was expressed in inclusion bodies in Escherichia coli at 25% of the total cellular protein content. Ion-exchange and gel-filtration chromatographies were applied to purify the chimaeric protein, achieving purity greater than 98%. We demonstrated that this chimaera can be expressed and purified in an active form; in vitro testing indicated that the chimaera fully retained the thrombolytic activity, platelet membrane-binding activity and anti-platelet aggregation activity of the parent molecules. The plasma clearance of the chimaera was similar to that of urokinase and ScuPA32k. In vivo experiments in a canine system indicated that animals administered the chimaera presented a decreased time to reperfusion, higher reperfusion ratio and less bleeding effects than treatment with urokinase. These results show that the chimaera is a platelet-targeted plasminogen activator with enhanced thrombolytic and antithrombotic potency that may have advantages over currently available thrombolytic agents.

Major histocompatibility (MHC) class I tetramers are used in the quantitative analysis of epitope peptide-specific CD8+ T-cells. An MHC class I tetramer was composed of 4 MHC class I complexes and a fluorescently labeled streptavidin (SA) molecule. Each MHC class I complex consists of an MHC heavy chain, a beta(2)-microglobulin (beta(2)m) molecule and a synthetic epitope peptide. In most previous studies, an MHC class I complex was formed in the refolding buffer with an expressed MHC heavy chain molecule and beta(2)m, respectively. This procedure inevitably resulted in the disadvantages of forming unwanted multimers and self-refolding products, and the purification of each kind of monomer was time-consuming. In the present study, the genes of a human/murine chimeric MHC heavy chain (HLA-A2 alpha1, HLA-A2 alpha2 and MHC-H2D alpha3) and beta(2)m were tandem-cloned into plasmid pET17b and expressed as a fusion protein. The recombinant fusion protein was refolded with each of the three HLA-A2 restricted peptides (HBc18-27 FLPSDFFPSI, HBx52-60 HLSLRGLPV, and HBx92-100 VLHKRTLGL) and thus three chimeric MHC class I complexes were obtained. Biotinylation was performed, and its level of efficiency was observed via a band-shift assay in non-reducing polyacrylamide gel electrophoresis (PAGE). Such chimeric MHC class I tetramers showed a sensitive binding activity in monitoring HLA/A2 restrictive cytotoxic T lymphocytes (CTLs) in immunized HLA/A*0201 transgenic mice.

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.

Mismatch repair (MMR) genes are among of the most important genes associated with colorectal cancer (CRC). Single-nucleotide polymorphisms (SNPs) are generally thought to provide important information across a wide spectrum of life sciences; however, no study of association between SNPs of MMR genes and Chinese sporadic colorectal cancer (SCRC) is available. We chose 29 reported single-nucleotide variants that have rarely been verified in a population-based study. We identified SNPs and the genotype–phenotype association in Chinese populations of 150 healthy individuals and 160 SCRC patients. We extracted the genomic DNA from the blood of these individuals and used sequencing to determine these SNPs. Three SNPs ( MLH1 394G→C, 655A→G, 1151T→A) occurred with a frequency of 8.8–11.2% in the Chinese population. These SNPs formed a series with combined effects. The haplotype of concurrent MLH1 655 and 1151 SNPs and the haplotype combinations of MLH1 1151, MLH1 394 occurred exclusively in SCRC. None of the other 26 variants were detected in the Chinese population.

The development of thrombolytic agent could provide invaluable progress for antithrombotic therapy. In this paper, we reported the cloning, purification and biochemical characterization of AnxB1ScuPAFap, a thrombus-ditargeting chimera composed of annexin B1, low molecular single-chain urokinase (ScuPA-32K) and fibrin-adherent peptide (dodecapeptide, Fap). In vitro test showed that, the chimera was a thrombolytic agent with anticoagulant activity and thrombus-ditargeting with the activated-platelet membrane binding and fibrin clot binding activity. Compared to urokinase, the chimera had less reperfusion time, higher reperfusion ratio, and less bleeding effects on coronary thrombolysis by clot lysis assay in dogs. Thus, the chimera appeared to be suitable for thrombolytic therapy of thrombus diseases.

The role of poly(D,L-lactic-co-glycolic acid, PLGA) microparticles on enhancing immune responses of multiepitope DNA vaccines was investigated in vitro and in vivo. pcDNA-SG encoding T and B cell epitopes of foot-and-mouth disease virus (FMDV) was encapsulated into PLGA microparticles. PLGA microparticles could protect themselves from nuclease degradation in vitro. PLGA-pcDNA-SG microparticles could be uptaken by cells and expressed His-tagged SG immunogen in vitro and in vivo. A prolonged expression and presentation of SG immunogen were observed by confocal laser scanning microscopy in the lymphocytes from the mice incubated with PLGA-pcDNA-SG microparticles, compared with the mice immunized with naked pcDNA-SG. PLGA-pcDNA-SG microparticles displayed a significant stronger immunogenicity than naked DNA vaccines with a higher titer of virus-specific antibody, elevated IFN-gamma production and enhanced lymphocyte proliferation. PLGA-DNA microparticle could elicit augmented humoral and cellular responses with reduced amounts and times of immunization.

To optimize previous candidate DNA vaccine, a cis-expression plasmid DNA encoding two genes, human IL-2 and multiple-epitopes genes of foot-and-mouth disease virus (FMDV) was constructed with internal ribosome entry site (IRES) from encephalomyocarditis virus (EMCV) and intramuscularly inoculated into mice at 1-week interval. Specific antibodies in serum and cytokines (IL-2, IL-4 and IFN-gamma) from splenocytes were detected by indirect ELISA. Splenocytes proliferation rate was determined by a standard 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) method. The results showed that higher specific antibody, proliferate rate and cytokines were induced by plasmid DNA cis-expression with IL-2 compared to non-cis-expression plasmid DNA. Another series of mice were inoculated with plasmid DNA and boosted with antigenic protein. Specific antibody, proliferation rate and cytokines were induced significantly higher than those of mice immunized with protein or plasmid DNA only. However, only the cis-expression plasmid DNA elicited higher neutralization antibody in mice and provided one third protection against homologous virus in guinea pigs. In conclusion, cis-expression strategy with IL-2 up-regulated specific immunological response and provide protection against homologous virus.

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.