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Articles by Sheng Xiong in JoVE

 JoVE Biology

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

1Department of Science, Borough of Manhattan Community College, City Universtiy of New York (CUNY), 2Department of Biology, Queens College, The City University of New York (CUNY), 3Biochemistry Program, The Graduate Center, Queens College, The City University of New York (CUNY)


JoVE 4373

We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-β signaling.

Other articles by Sheng Xiong on PubMed

[Re-cloning of THP Gene and Construction of High Efficient Expression Yector of Volvariella Volvacea]

PCR technique was used for amplifying THP gene in an unknown vector with primer AFP1 and AFP2. Then THP gene was ligated to pGEM T-Vector to be the plasmid pGTHP4. The plasmid pCAMBIA1301 was digested with restriction enzyme BstE II and Nco I, and digestion product was separated with 1% of agarose gel, then big fragment containing promoter was isolated and purified with the Agarose Gel DNA Extraction Kit. At the same way, the plasmid pGTHP4 was digested with restriction enzyme BstZ I and Nco I, and the small fragment containing THP gene was purified from 1% agarose gel with the Agarose Gel DNA Extraction Kit. The big fragment and the small fragment were ligated at Nco I digested cohesive-end. The ligation product was re-ligated to be cyclic plasmid by addition to a specific adapter, resulting in the pCTH823, a expression vectorof V. volvacea.

[Influence of the Reductase Deficient Escherichia Coli on the Solubility of Recombinant Proteins Produced in It]

The cytoplasm of E. coli is a reducing environment where cysteines do not engage in disulfide bonds. Any disulfide bonds that do appear are rapidly reduced through the action of disulfide reducing enzymes such as thioredoxin and glutaredoxin. To study the influence of E. coli cytoplasm on the solubility of recombinant proteins produced in it, bovine fibroblast growth factor (BbFGF), with single disulfide bond, and anti-HBsAg single-chain Fv (HBscFv), with two disulfide bonds, were selected as the pattern molecules of simple protein and complex protein, respectively. pJN98-BbFGF, a BbFGF expressing plasmid based on the vector pET3c, was constructed and transformed into normal host BL21(DE3) and a reductase deficient strain, E. coli Origami(DE3). At the same time, pQE-HBscFv, a HBscFv expressing plasmid was constructed and transformed into M15 [pREP4] and Origami(DE3). The recombinant BbFGF and HBscFv were produced in 2 types of bacteria and their solubilities and bioactivities were determined, respectively. It was found that the majority of BbFGF had formed inclusion body in the cytoplasm of BL21 (DE3) and all of them turned into soluble protein in Origami(DE3). It was also found the productivity of BbFGF in Origami (DE3) was 5% - 10% of the total protein and the value was 15% - 23% in BL21(DE3). BbFGFs produced in 2 recombinant bacteria were purified by cation exchange and heparin affinity chromatography. MTT assay revealed that the bioactivity of BbFGF purified from Origami(DE3) was higher than its counterpart from BL21(DE3). The ED50 of BbFGFs from different bacteria was 1.6ng/mL and 2.2ng/mL, respectively. As far as HBscFvs, both of them formed inclusion body in the cytoplasm of M15 [pQE-HBscFv] and Origami [pQE-HBscFv]. The inclusion body was solubilized in 6mol/L GuHCl, purified with a His-Trap column and then refolded by dialysis step-by-step against buffers containing downtrend concentration of GuHCl. Indirect ELISA was applied to determine the HBsAg binding activity of HBscFvs. It was found there was no obvious difference between the bioactivity of refolded HBscFvs produced from 2 recombinant bacteria. On the other hand, the supernatant of Origami [pQE-HBscFv] lysate displayed weak bioactivity and its counterpart from M15 [pQE-HBscFv] displayed without any bioactivity. The soluble HBsFv in the cytoplasm of Origami [pQE-HBscFv] was purified by cation exchange and immobilized metal affinity chromatography (IMAC) and the yield was 1 - 2mg/L. Those results suggested that modification of the redox environment of E. coli cytoplasm greatly improved the solubility of recombinant disulfide-bonded proteins produced in it. In the next step, we had like to co-express of molecular chaperones or refoldase to raise the yield of soluble recombinant proteins, as well as optimizing the culture condition of the "oxidizing" E. coli.

[Expression of a Human Single-chain Fv Antibody Against HBsAg in Pichia Pastoris]

To express and secrete native HBscFv (anti-HBsAg single-chain Fv) in P. pastoris, HBscFv was amplified from plasmid pGEM-HBscFv, and then sub-cloned into expression vector pPICZalphaA. The resulting plasmid pPIC-HBscFv was linearized and transformed into P. pastoris GS115. The recombinant Pichia strains, identified by direct PCR and Zeocin-resistant screening of Pichia transformants, were cultured and induced with methanol. It was found that recombinant HBscFv, lead by alpha-factor, could be secreted into the culture supernatant to a level of 80mg/L. The bioactivity of Pichia produced HBscFv was confirmed by indirect ELISA, which also suggested that the bioactivity of HBscFv in the culture supernatant reached its peak in 72h and decreased in the late-stage of the induction. PAS staining suggests that HBscFv produced by yeast is poorly glycosylated or none-glycosylated protein.

[Physical and Chemical Characters of Recombinant Human Nucleoside Diphosphate Kinase A]

To purify recombinant human nucleoside diphosphate kinase A (rhNDPK-A) and determine its physical and chemical characters, recombinant NDPK-A producing E. coli was cultured in 80L fermentor under high cell density culture (HCDC) conditions. The harvested cells were treated with high pressure to break the cell up, tangential-flow microfiltration to remove the bacteria debris and ultrafiltration to concentrate the filtered solution containing target protein. The crude NDPK-A was purified by ion exchange chromatography with DEAE Sepharose Fast Flow, affinity chromatography with Cibarcron Blue 3GA Sepharose CL-4B and gel filtration with Sephadex G-100. The purity of rhNDPK-A was analyzed with SDS-PAGE and RP-HPLC. The Enzymatic activity was determined with RP-HPLC. The molecular weight (MW) was measured with matrix assisted laser desorption ionization time-of-flight MS (MALDI-TOF MS). The N-terminal residue was sequenced with Edman method. The apparent molecular weight of rhNDPK-A in solution was determined with multiangle laser light-scattering method (MALS). It was found that the purity of rhNDPK-A was 97.3% with SDS-PAGE method and 99.2% with RP-HPLC method. The specific enzymatic activity was (900 +/- 100) u/mg. The molecular weight was 17017, which was 132 less than the calculated value according to the amino acid sequence of NDPK-A. The sequencing result of rhNDPK-A revealed that its N-terminal residue was Ala, which was the second residue on N-terminal of native NDPK-A. The calculated MW of N-terminal deleted rhNDPK-A was 17017, exactly equal to the experimental value. The result of apparent MW determination revealed that rhNDPK-A formed homohexamer in solution with a MW of 102kD. These results suggested that rhNDPK-A possessed character identical to its native counterpart of assembling into hexamer. Confirming the identity of rhNDPK-A to its native counterpart provided a good foundation for drug development and mechanism study of NDPK-A.

[Production of Recombinant Humanized Anti-HBsAg Fab Antibody by Fermentation]

In order to produce recombinant human anti-HBsAg Fab antibody in Pichia pastoris, the recombinant yeast was fermented using fed-batch system in a 30 L bioreactor. The fermentation temperature was 30 degrees C, the pH was 5.0 approximately 5.3, and the DO was 20% approximately 30%. The recombinant Fab antibody was purified from crude culture supernatant by ion exchange and analyzed by SDS-PAGE and western blot and ELISA. When the absorbance (OD600) of broth reach 300 at the end of fed-batch phase, the induced phase was initiated. The results showed that recombinant human anti-HBsAg Fab antibody was high-level expressed in recombinant Pichia pastoris using a fed-batch fermentation system. Both chains of the Fab were successfully expressed upon methanol induction. After 192 h of induction, the expression level of recombinant Fab (soluble) reached 412 mg/L. The recombinant Fab antibody was purified effectively by ion-exchange chromatography from the fermentation supernatant to a purity of 95%. And the affinity activities of the purified recombinant Fab antibdy and fermentation supernatant were detected, and both of them showed high affinity activities. The results demonstrated that recombinant human anti-HBsAg Fab antibody could be high level produced by fed-batch fermentations in Pichia pastoris. Which can be efficiently used in industrial production.

Immunogenicity of SARS Inactivated Vaccine in BALB/c Mice

Severe acute respiratory syndrome (SARS) is a serious infectious threat to public health. To create a novel trial vaccine and evaluate its potency, we attempted to generate a SARS inactivated vaccine using SARS coronavirus (SARS-CoV) strain F69 treated with formaldehyde and mixed with Al(OH)3. Three doses of the vaccine were used to challenge three groups of BALB/c mice. We found that the mice exhibited specific IgM on day 4 and IgG on day 8. The peak titers of IgG were at day 47 in low-dose group (1:19,200) and high-dose group (1:38,400) whereas in middle-dose group (1:19,200), the peak was at day 40. On day 63, the IgG levels reached a plateau. Neutralization assay demonstrated that the antisera could protect Vero-E6 cells from SARS-CoV's infection. Analysis of the antibody specificity revealed that the mouse antisera contained a mixture of antibodies specifically against the structure proteins of SARS-CoV. Furthermore, the mouse antisera conferred higher amount of antibodies against protein N, polypeptide S4 and S2 than those of proteins M and 3CL. These findings suggest that the inactivated SARS-CoV could preserve its antigenicity and the inactivated vaccine can stimulate mice to produce high levels of antibodies with neutralization activity. Results also suggest that polypeptides originating from protein N or S might be a potential target for the generation of a recombinant SARS vaccine.

[Preparation and Characterization of Monoclonal Antibodies Against Recombinant Human NDPK-A]

To prepare monoclonal antibodies (mAb) against recombinant human nucleoside diphosphate kinase-A(NDPK-A) and characterize their properties.

Construction of a Eukaryotic Expression Plasmid Encoding Partial S Gene Fragments of the SARS-CoV and Its Potential Utility As a DNA Vaccine

The spike (S) protein, a main surface antigen of the SARS coronavirus (SARS-CoV), is considered to be one of the most important protective antigen candidates for targets for vaccine design against the virus. In this study, a secreted recombinant expression plasmid, pVAX-S1, encoding the partial S protein with a signal peptide, was constructed and used to immunize BALB/c mice through electroporation. It was demonstrated that the eukaryotic expression vector pVAX-S1 was successfully constructed by restriction enzyme and sequence analysis. The expressed protein could be detected specifically by Western blot analysis. The serum IgG level of the vaccine group mice was significantly higher than that of the corresponding control group at day 14 after vaccination (P < 0.05). The vaccine group demonstrated significantly higher S1 protein lymphocyte proliferation index (LPI) than the control groups (P < 0.05). Furthermore, in the experimental group, a decrease in the ratio of CD4(+) to CD8(+) T-lymphocytes and an increase level of IFN-gamma in serum were observed. However, interleukin-4 (IL-4) was not detectable in two groups. These results strongly demonstrated that the pVAX-S1 plasmid could induce humoral and cellular immune responses in mice, and may be a potential candidate for a DNA vaccine against the SARS coronavirus.

Production of Recombinant Humanized Anti-HBsAg Fab Fragment from Pichia Pastoris by Fermentation

In this report, we describe the high-yield secretory expression of the recombinant human anti-HBsAg Fab fragment from Pichia pastoris that was achieved by co-integration of the genes encoding the heavy and light chains (both under the control of alcohol oxidase promoter) into the genome of the yeast cells. The fed-batch fermentations were carried out in a 5 L scale. Both chains of the Fab were successfully expressed upon methanol induction. The absorbance (OD600) of the broth can reach 350 approximately 500 at the end of fed-batch phase. After the induction, the expression level of the recombinant Fab (soluble) reached 420 approximately 458 mg/L. The recombinant Fab fragment was purified from the crude culture supernatant by ion exchange chromatography and the purity of the recombinant Fab fragment was over 95%. The affinity activities of the crude fermentation supernatant and the purified Fab were analyzed by indirect ELISA, which showed that the purified recombinant Fab fragment had high affinity activity with hepatitis B surface antigen.

[Transformation of Volvariella Volvacea with a Thermal Hysteresis Protein Gene by Particle Bombardment]

A cDNA encoding a thermal hysteresis protein was isolated from the Swedish Arctic insect spruce budworm by RT-PCR amplification. Volvariella volvacea strain V34 was transformed with this cDNA through particle bombardment. PCR detection and Southern blotting analysis show that the thermal hysteresis protein gene is integrated into Volvariella volvacea genome. Cold stress assay reveals that transgenic Volvariella volvacea lines exhibit stronger cold tolerance than host strain. The morphological observation of transgenic Volvariella volvacea lines shows that growth rates of most Volvariella volvacea transformants are significantly slower than that of negative control strain. And hypha of most Volvariella volvacea tansformants is thinner than host strain's hypha. Transformant screening result indicates that three-round of selection procedure with first selection on PDSA solid selective medium followed by second and third selection in PDSB liquid selective medium is favorable to get genuine transformants and to eliminate false transformants. Cold tolerance assay of transgenic Volvariella volvacea F1 generation demonstrates that the progeny of transgenic Volvariella volvacea still possesses stronger cold tolerance than non-transformed host strain. This suggests that the cold tolerant characteristic of transgenic Volvariella volvacea is meiotically stable between generations.

Immune Responses in Balb/c Mice Induced by a Candidate SARS-CoV Inactivated Vaccine Prepared from F69 Strain

The immunogenicity of a candidate-inactivated vaccine prepared from SARS-CoV F69 strain was evaluated in Balb/c mice. Potent humoral immune responses were induced under the elicitation of three times of immunizations at 2-week intervals with this vaccine, combined with three types of adjuvants (Freund's adjuvant, Al(OH)(3) adjuvant and CpG adjuvant). Titers of specific IgG antibodies in three test groups all peaked in the sixth week after first vaccination, but significant differences existed in the kinetics of specific IgG antibody levels. The strong neutralizing capacity exhibited in micro-cytopathic effect neutralization tests indicated the specific antibodies are protective. Western blot assay further demonstrated the specificity of the induced serum antibodies.

Antiviral Activity of Cepharanthine Against Severe Acute Respiratory Syndrome Coronavirus in Vitro

Solubility of Disulfide-bonded Proteins in the Cytoplasm of Escherichia Coli and Its "oxidizing" Mutant

To study the influence of redox environment of Escherichia coli (E. coli) cytoplasm on disulfide bond formation of recombinant proteins.

[Analysis of the Relationship Between Nm23-H1 Gene and Human Chronic Myeloblastic Leukemia Using SiRNA]

To investigate the relationship between nm23-H1 gene and human chronic myeloblastic leukemia we designed siRNAs which target nm23-H1 gene. According to the principles of designing siRNA, we selected three siRNAs and transfected them into K562 cells by lipofectamine2000. The expression levels of nm23-H1 mRNA were detected by reverse transcriptase polymerase chain reaction after transfection for 24 hours. The expression levels of nm23-H1 protein were assayed by immunocytochemical method after transfection for 48 hours. And after transfection for 24, 48 and 72 hours, cell proliferation was determined by MTT method. Among the three siRNAs, siNM526 can effectively inhibit the expression of nm23-H1 on mRNA and protein levels. The growth of K562 cells was suppressed after transfection of siNM526. These results suggest that low expression level of nm23-H1 in K562 cells inhibited cell proliferation, namely reduced malignant degree of them. Therefore nm23-H1 gene might be a potential target of leukemia treatment.

[Efficient Purification of Recombinant Human NDPK-A in Pilot-scale]

To purify recombinant human nucleoside diphosphate kinase A (rhNDPK-A) efficiently in pilot scale, cells of rhNDPK-A producing E. coli were homogenized by high pressure under 4 degrees C, 950 Pa. The insoluble debris was removed by microfiltration and the soluble portion was concentrated by ultrafiltration. The resulted crude sample was loaded on DEAE-sepharose Fast Flow. The target fraction was collected and then load on Cibacron Blue 3GA Sepharose CL-4B. Eluted with buffer containing ATP from the AC column, rhNDPK-A was polished with ultrafiltration. The results showed that after homogenized 2 rounds, 1500g cells of E. coli brought crude sample containing 47.6g NDPK-A. Treated with microfiltration and ultrafiltration, 27.3g of NDPK-A were recovered from this bacteria homogenate. After 2-step purification with column chromatography and then polished with ultrafiltration, 17.2 g rhNDPK-A were collected with purity of 96.3%. The recovery of the whole purification process was 36.2%, and the productivity of rhNDPK-A was 1.15 g per 100 g wet cells. Comparing the recovery of each purification step, it was found that the recovery of polish is higher than that of affinity chromatography, which is higher than that of ion exchange chromatography. The limit step was the process of sample pretreatment among the 4 purification steps. Combine with the fermentation results reported before, it was deduced that the productivity of rhNDPK-A was 510 mg/L. In conclusion, an easily controlled purification condition with high yield provides material for the translation researches of NDPK; In addition, it was suggested the crucial step determine the recovery of non-secretive recombinant proteins might be the process of sample pretreatment, not be the process of column chromatography.

[Influence of Nm23-H1 Gene Silence in K562 Cell on Its Differentiation Toward Megakaryocyte]

To construct a stable nm23-H1-knock-down cell model with K562 cell line and study its differentiation toward megakaryocyte.

High-throughput Assay Using a GFP-expressing Replicon for SARS-CoV Drug Discovery

The causative agent of severe acute respiratory syndrome (SARS) has been identified as a novel coronavirus, SARS-CoV. The development of rapid screening assays is essential for antiviral drug discovery. By using a cell line expressing a SARS-CoV subgenomic replicon, we developed a high-throughput assay and used it to screen small molecule compounds for inhibitors of SARS-CoV replication in the absence of live virus. The assay system involves minimal manipulation after assay set-up, facilitates automated read-out and minimizes risks associated with hazardous viruses. Based on this assay system, we screened 7035 small molecule compounds from which we identified 7 compounds with anti-SARS-CoV activity. We demonstrate that the compounds inhibited SARS-CoV replication-dependent GFP expression in the replicon cells and reduced SARS-CoV viral protein accumulation and viral RNA copy number in the replicon cells. In a SARS-CoV plaque reduction assay, these compounds were confirmed to have antiviral activity. The target of one of the hit compounds, C12344, was validated by the generation of resistant replicon cells and the identification of the mutations conferring the resistant phenotype. These compounds should be valuable for developing anti-SARS therapeutic drugs as well as research tools to study the mechanism of SARS-CoV replication.

Fractionation of Proteins by Heparin Chromatography

Heparins are negatively charged polydispersed linear polysaccharides which have the ability to bind a wide range of biomolecules including enzymes, serine protease inhibitors, growth factors, extracellular matrix proteins, DNA modification enzymes and hormone receptors. In this chromatography, heparin is not only an affinity ligand but also an ion exchanger with high charge density and distribution. Heparin chromatography is an adsorption chromatography in which biomolecules can be specifically and reversibly adsorbed by heparins immobilized on an insoluble support. An advantage of this chromatography is that heparin-binding proteins can be conveniently enriched using its concentration effect. This is especially important for separating low abundance proteins for the analysis in two-dimensional electrophoresis (2DE) or other proteomics approaches. Heparin chromatography is a powerful sample-pretreatment technology that has been widely used to fractionate proteins from extracts of prokaryotic organism or eukaryotic cells. As an example, the fractionation of fibroblast growth factors (FGFs) from the extract of mouse brain microvascular endothelial cells (MVEC) is now introduced to demonstrate the procedure of heparin chromatography.

Comparative Proteomic Analysis to Discover Potential Therapeutic Targets in Human Multiple Myeloma

To clarify the molecular mechanisms that participate in the formation of multiple myeloma (MM) and to detect any tumor-related biomarkers, we performed proteomic analysis of cellular protein extracts from MM cells and normal plasma cells. Plasma cells from nine patients with newly diagnosed MM and nine healthy donors were purified by using anti-CD138 based immunomagnetic bead-positive selection. The protein profiles of purified MM and normal plasma cells were compared using 2-DE. We identified a total of 43 differentially expressed proteins, and confirmed with Western blotting six proteins. The altered proteins were analyzed using the software program Pathway Studio and the biological network can be accessed via (http://life-health.jnu.edu.cn/pathway/pathway.html). Further functional studies showed that annexin A1 knock down modestly induces lethality alone and potentiates the effects of dexamethasone on both dexamethasone-sensitive and dexamethasone-resistant MM cells. By correlating the proteomic data with these functional studies, the current results provide not only new insights into the pathogenesis of MM but also direct implications for the development of novel anti-MM therapeutic strategies and could lead to the discovery of potential therapeutic targets. Future molecular and functional studies would provide novel insights into the roles of these dysregulated proteins in the molecular etiology of MM.

Transcriptomic and Proteomic Approach to Studying SNX-2112-induced K562 Cells Apoptosis and Anti-leukemia Activity in K562-NOD/SCID Mice

SNX-2112, a novel inhibitor of Hsp90 currently used as an anti-tumor drug, induces apoptosis in multiple tumor cell lines. It destabilizes specific client proteins, but the molecular mechanism of the apoptosis effect of SNX-2112 is poorly understood. Here, we analyzed the apoptotic effect of SNX-2112 on human chronic myeloid leukemia (CML) K562 cells. Transcriptomic and proteomic approaches further revealed that caspase signals originated from mitochondria dysfunction, mediated by Akt signaling pathway inactivity. Additionally, SNX-2112 prolonged the survival time of NOD/SCID mice inoculated with K562 tumor cells. Our results demonstrated the therapeutic potential of SNX-2112 against human CML.

Proteomic and Functional Analyses Reveal a Dual Molecular Mechanism Underlying Arsenic-induced Apoptosis in Human Multiple Myeloma Cells

Multiple myeloma (MM) is an incurable plasma cell malignancy with a terminal phase marked by increased proliferation and resistance to therapy. Arsenic trioxide (ATO), an antitumor agent with a multifaceted mechanism of action, displayed clinical activity in patients with late-stage multiple myeloma. However, the precise mechanism(s) of action of ATO has not been completely elucidated. In the present study, we used proteomics to analyze the ATO-induced protein alterations in MM cell line U266 and then investigated the molecular pathways responsible for the anticancer actions of ATO. Several clusters of proteins altered in expression in U266 cells upon ATO treatment were identified, including down-regulated signal transduction proteins and ubiquitin/proteasome members, and up-regulated immunity and defense proteins. Significantly regulated 14-3-3zeta and heat shock proteins (HSPs) were selected for further functional studies. Overexpression of 14-3-3zeta in MM cells attenuated ATO-induced cell death, whereas RNAi-based 14-3-3zeta knock-down or the inhibition of HSP90 enhanced tumor cell sensitivity to the ATO induction. These observations implicate 14-3-3zeta and HSP90 as potential molecular targets for drug intervention of multiple myeloma and thus improve our understanding on the mechanisms of antitumor activity of ATO.

Nm23-H1 Regulates the Proliferation and Differentiation of the Human Chronic Myeloid Leukemia K562 Cell Line: a Functional Proteomics Study

Nm23-H1 is a suppressor of metastasis that has been implicated in the regulation of proliferation and differentiation of hematopoietic cells, although specific mechanisms for Nm23-H1 have not been well-characterized. Our study is designed to further elucidate the role of Nm23-H1 in the human chronic myeloid leukemia K562 cell line.

Quantitative Phosphoproteomics of Proteasome Inhibition in Multiple Myeloma Cells

The proteasome inhibitor bortezomib represents an important advance in the treatment of multiple myeloma (MM). Bortezomib inhibits the activity of the 26S proteasome and induces cell death in a variety of tumor cells; however, the mechanism of cytotoxicity is not well understood.

[Expression, Purification and Activity Determination of Cyanovirin-N]

Cyanovirin-N (CVN) is an 11 kDa anti-HIV protein originally isolated from extracts of a cyanobacterium, Nostoc ellipsosporum. The protein binds with high affinity to the viral envelope glycoprotein gp120 and irreversibly inactivates diverse HIV strains. A fusion gene consisting of cvn, sumo and 6xHis tag was synthesized by PCR according to the codon bias of Escherichia coli. The fusion protein is expressed in the cytoplasm of E. coli in a soluble form and up to 28% of the total protein. The recombinant CVN was purified to homogeneity by 2 rounds of Ni-NTA affinity chromatography and one round of SUMO protease cleavage. Bioactivity assay demonstrated that SUMO-CVN and CVN bound to gp120 with nanomolar concentration. In addition, CVN showed potent anti-HSV-1 and anti-HIV-1 activities in in vitro cellular assays. Therefore, the 6xHis SUMO fusion expression and purification system provides a better approach for large scale production of CVN for further microbicide development.

A Cys/Ser Mutation of NDPK-A Stabilizes Its Oligomerization State and Enhances Its Activity

Nucleoside diphosphate phosphate transferase A (NDPK-A) has been shown to play critical roles in the regulation of proliferation, differentiation, growth and apoptosis of cells. Our previous study suggested that the disulphide cross-linkage between cysteine 4 (C4) and cysteine 145 (C145) of NDPK-A might be a possible regulator of its activity. To confirm this hypothesis, the C145 residue of NDPK-A was mutated to serine, and the isomerization and biological activities of the mutant were investigated and compared with those of its wild-type counterpart. It was found the C145S mutation eliminated the intramolecular disulphide bond (DB) and prevented the formation of intermolecular DB, which was known to dissociate the hexameric NDPK-A into dimeric one. We also demonstrated that the C145S mutation didn't affect the autologous hexamerization of this protein, and the mutant had increased bioactivities including phosphate transferase and DNase. These findings support the hypothesis that the formation of DBs in NDPK-A is involved in the regulation of the oligomerization and bioactivity of this multiple function protein, and that C145 is a key residue in the regulation of NDPK-A. In addition, the C145S mutant that we have constructed might be an attractive candidate for use in applications that require NDPK-A.

Proteomic Analysis Reveals Novel Binding Partners of MIP-T3 in Human Cells

MIP-T3 (microtubule-interacting protein associated with TRAF3) is a microtubule-interacting protein that evolutionarily conserved from worms to humans, but whose cellular functions remains unknown. To get insight into the functions of MIP-T3, we set out to identify MIP-T3 interacting proteins by immunoprecipitation in human embryonic kidney 293 cells and MS analysis. As the results, a total of 34 proteins were identified and most of them were novel MIP-T3 putative partners. The MIP-T3-associated proteins could be grouped into nine clusters based on their molecule functions, including cytoskeleton, chaperone, nucleic acid binding, kinase and so on. Three MIP-T3-interacted proteins - actin, HSPA8 and tubulin - were further confirmed by reciprocal coimmunoprecipitations and colocalization analysis. The interaction of MIP-T3 with both actin filaments and microtubule suggested that MIP-T3 may play an important role in regulation of cytoskeleton dynamics in cells. Our results therefore not only uncover a large number of MIP-T3-associated proteins that possess a variety of cellular functions, but also provide new research directions for the study of the functions of MIP-T3.

Transient Expression of Recombinant SPDGFR Alpha-Fc in CHO DG44 Cells Using 50-mL Orbitally Shaking Disposable Bioreactors

Overactivity of platelet-derived growth factor (PDGF) has been linked to malignant cancers. High levels of PDGF result in the activation of its receptors (PDGFRs) and the over-proliferation of cells. Therefore, interfering with this signaling pathway in cancer cells could be significant for anti-cancer drug development. In a previous study, the sPDGFR alpha-Fc fusion protein expressed in static CHO-k(1) cells showed an anti-proliferative effect on vascular endothelial cells. However, it was difficult to obtain a large quantity of this fusion protein for further functional studies. In the present study, the sPDGFR alpha-Fc fusion protein was transiently expressed in Chinese Hamster Ovary (CHO) DG44 cells in 50-mL orbital shaking bioreactors. sPDGFR alpha-Fc was expressed as a 250-kDa dimeric protein with potential glycosylation. The final yield of sPDGFR alpha-Fc in the culture supernatant was as high as 16.68 mg/L. Our results suggest that transient expression in orbital shaking bioreactors may be feasible for preparation of recombinant proteins used for preclinical studies.

The Antiviral Protein Cyanovirin-N: the Current State of Its Production and Applications

Human immunodeficiency virus (HIV)/AIDS continues to spread worldwide, and most of the HIV-infected people living in developing countries have little or no access to highly active antiretroviral therapy. The development of efficient and low-cost microbicides to prevent sexual transmission of HIV should be given high priority because there is no vaccine available yet. Cyanovirin-N (CVN) is an entry inhibitor of HIV and many other viruses, and it represents a new generation of microbicide that has specific and potent activity, a different mechanism of action, and unusual chemicophysical stability. In vitro and in vivo antiviral tests suggested that the anti-HIV effect of CVN is stronger than a well-known gp120-targeted antibody (2G12) and another microbicide candidate, PRO2000. CVN is a cyanobacteria-derived protein that has special structural features, making the artificial production of this protein very difficult. In order to develop an efficient and relatively low-cost approach for large-scale production of recombinant CVN to satisfy medical use, this protein has been expressed in many systems by trial and error. Here, to summarize the potential and remaining challenges for the development of this protein into an HIV prevention agent, the progress in the structural mechanism determination, heterologous production and pharmacological evaluation of CVN is reviewed.

In Vitro and in Vivo Antineoplastic Activity of a Novel Bromopyrrole and Its Potential Mechanism of Action

Many bromopyrrole compounds have been reported to have in vitro antineoplastic activity. In a previous study, we isolated N-(4, 5-dibromo-pyrrole-2-carbonyl)-L-amino isovaleric acid methyl ester (B6) from marine sponges. Here, we investigated the in vitro and in vivo antineoplastic activity of B6 and its potential mechanism.

Soluble Cytoplasmic Expression, Rapid Purification, and Characterization of Cyanovirin-N As a His-SUMO Fusion

Cyanovirin-N (CVN) is a promising antiviral candidate that has an extremely low sequence homology with any other known proteins. The efficient and soluble expression of biologically functional recombinant CVN (rCVN) is still an obstacle due to insufficient yield, aggregation, and abnormal modification. Here, we describe an improved approach to preparing native rCVN from Escherichia coli more efficiently. A fusion gene consisting of cvn and sumo (small ubiquitin-related modifier) and a hexahistidine tag was constructed according to the codon bias of the host cell. This small ubiquitin-related modifier (SUMO)-fused CVN is expressed in the cytoplasm of E. coli in a folded and soluble form (>30% of the total soluble protein), yielding 3 to 4 mg of native rCVN from 1 g of wet cells to a purity up to 97.6%. Matrix-assisted laser desorption ionization coupled to time-of-flight mass spectrometry and reverse-phase high-performance liquid chromatographic analysis showed that the purified rCVN was an intact and homogeneous protein with a molecular weight of 11,016.68 Da. Potent antiviral activity of rCVN against herpes simplex virus type 1 and human immunodeficiency virus type 1/IIIB was confirmed in a dose-dependent manner at nanomolar concentrations. Thus, the His-SUMO double-fused CVN provides an efficient approach for the soluble expression of rCVN in the cytoplasm of E. coli, allowing an alternative system to develop bioprocess for the large-scale production of this antiviral candidate.

Cloning and Characterization of Porcine 4Ig-B7-H3: a Potent Inhibitor of Porcine T-cell Activation

Members of the B7 superfamily costimulate the proliferation of lymphocytes during the initiation and maintenance of antigen-specific humoral and cell-mediated immune responses. B7-H3 (CD276) is a newly identified member of the B7 superfamily. It has been shown that B7-H3 plays a significant role in regulating T cell response in humans and mice, but it is not known whether a counterpart of human or murine B7-H3 exists in porcine species.

On-line Monitoring of Oxygen in Tubespin, a Novel, Small-scale Disposable Bioreactor

A novel, optical sensor was fixed in a new type of disposable bioreactor, Tubespin, for the on-line (real-time) monitoring of dissolved oxygen concentrations during cell culture. The cell density, viability and volumetric mass transfer coefficient were also determined to further characterize the bioreactors. The k(L)a value of the Tubespin at standard conditions was 24.3 h(-1), while that of a spinner flask was only 2.7 h(-1). The maximum cell density in the Tubespin bioreactor reached 6 × 10(6) cells mL(-1), which was two times higher than the cell density in a spinner flask. Furthermore, the dynamic dissolved oxygen level was maintained above 90% air-saturation in the Tubespin, while the value was only 1.9% in a spinner flask. These results demonstrate the competitive advantage of using the Tubespin system over spinner flasks for process optimization and scale-down studies of oxygen transfer and cell growth.

[Purification of Cyanovirin-N with Antiviral Activity and Preparation As Well As Modification of Its Polyclonal Antibody]

To purify the recombinant Cyanovirin-N (CVN) and determine its anti-influenza virus A (H1N1) activity, and to prepare the polyclonal antibody of CVN, purify it and label it with an enzyme for future application.

The C-terminus of MIP-T3 Protein is Required for Ubiquitin-proteasome-mediated Degradation in Human Cells

The intraflagellar transport (IFT) complex is essential for the formation and functional maintenance of eukaryotic cilia which play a vital role in development and tissue homeostasis. However, the biochemical characteristics and precise functions of IFT proteins remain unknown. Here, we report that MIP-T3, a human microtubule-interacting protein recently identified as a novel conserved component of the IFT complex, is an easily degradable protein in human cell lines. Protein degradation is mediated by the ubiquitin-proteasome system, and the C-terminus is required for ubiquitination and proteasome-mediated degradation of MIP-T3 protein. This study provides the first evidence for regulation of IFT protein stability.

Antiviral Activity and Possible Mechanisms of Action of Pentagalloylglucose (PGG) Against Influenza A Virus

Influenza A virus (IAV) infection is a major public health threat leading to significant morbidity and mortality. The emergence of drug-resistant virus strains highlights the urgent need to develop novel antiviral drugs with alternative modes of action. Pentagalloylglucose (PGG), a naturally occurring polyphenolic compound, possesses a broad spectrum of biological activities. In this study, we found that PGG has anti-influenza-virus activity, and investigated its possible mechanism(s) of action in vitro. Both pre-incubation of virus prior to infection and post-exposure of infected cells with PGG significantly inhibited virus yields. Influenza-virus-induced hemagglutination of chicken red blood cells was inhibited by PGG treatment, suggesting that PGG can inhibit IAV infection by interacting with the viral hemagglutinin. PGG did not affect viral protein synthesis or nuclear transport of viral nucleoprotein (NP) but greatly reduced plasma membrane accumulation of NP protein at the late stage of the replication cycle. Furthermore, PGG significantly reduced virus budding and progeny virus release from infected cells. This study revealed for the first time that PGG can inhibit IAV replication with a dual mode of action and offers new insights into its underlying mechanisms of antiviral action.

C. Elegans ADAMTS ADT-2 Regulates Body Size by Modulating TGFβ Signaling and Cuticle Collagen Organization

Organismal growth and body size are influenced by both genetic and environmental factors. We have utilized the strong molecular genetic techniques available in the nematode Caenorhabditis elegans to identify genetic determinants of body size. In C. elegans, DBL-1, a member of the conserved family of secreted growth factors known as the Transforming Growth Factor β superfamily, is known to play a major role in growth control. The mechanisms by which other determinants of body size function, however, is less well understood. To identify additional genes involved in body size regulation, a genetic screen for small mutants was previously performed. One of the genes identified in that screen was sma-21. We now demonstrate that sma-21 encodes ADT-2, a member of the ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family of secreted metalloproteases. ADAMTS proteins are believed to remodel the extracellular matrix and may modulate the activity of extracellular signals. Genetic interactions suggest that ADT-2 acts in parallel with or in multiple size regulatory pathways. We demonstrate that ADT-2 is required for normal levels of expression of a DBL-1-responsive transcriptional reporter. We further demonstrate that adt-2 regulatory sequences drive expression in glial-like and vulval cells, and that ADT-2 activity is required for normal cuticle collagen fibril organization. We therefore propose that ADT-2 regulates body size both by modulating TGFβ signaling activity and by maintaining normal cuticle structure.

In Vitro Anti-herpes Simplex Virus Activity of 1,2,4,6-tetra-O-galloyl-β-D-glucose from Phyllanthus Emblica L. (Euphorbiaceae)

In this study, 1,2,4,6-tetra-O-galloyl-β-D-glucose (1246TGG), a polyphenolic compound isolated from traditional Chinese medicine Phyllanthus emblica L. (Euphorbiaceae), was found to inhibit herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) infection at different magnitudes of activity in vitro. Further studies revealed that 1246TGG directly inactivated HSV-1 particles, leading to the failure of early infection, including viral attachment and penetration. 1246TGG also suppressed the intracellular growth of HSV-1 within a long period post-infection (from 0 h p.i. to 12 h p.i.), while it might exert an antiviral effect mainly before 3 h p.i. It inhibited HSV-1 E and L gene expressions as well as viral DNA replication but did not affect the RNA synthesis of IE gene in our study. Also, in the presence of 1246TGG, the synthesis of viral protein was reduced. Taken together, it was suggested that 1246TGG might exert anti-HSV activity both by inactivating extracellular viral particles and by inhibiting viral biosynthesis in host cells. These results warrant further studies on the antiviral mechanisms of 1246TGG and suggest that it might be a candidate for HSV therapy.

Pentagalloylglucose Downregulates Cofilin1 and Inhibits HSV-1 Infection

To investigate the anti-herpesvirus mechanism of pentagalloylglucose (PGG), we compared the proteomic changes between herpes simplex virus type 1 (HSV-1) infected MRC-5 cells with or without PGG-treatment, and between non-infected MRC-5 cells with or without PGG-treatment by 2-DE and MS-based analysis. Differentially expressed cellular proteins were mainly involved with actin cytoskeleton regulation. Significantly, PGG can down-regulate cofilin1, a key regulator of actin cytoskeleton dynamics. PGG can inhibit HSV-1-induced rearrangements of actin cytoskeleton which is important for infectivity. Furthermore, cofilin1 knockdown by siRNA also inhibited the HSV-1-induced actin-skeleton rearrangements. Both PGG-treatment and cofilin1 knockdown can reduce HSV-1 DNA, mRNA, protein synthesis and virus yields. Altogether, the results suggested that down-regulating cofilin1 plays a role in PGG inhibiting HSV-1 infection. PGG may be a promising anti-herpesvirus agent for drug development.

Role of Binding and Nucleoside Diphosphate Kinase A in the Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator by AMP-activated Protein Kinase

Cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel mutations cause cystic fibrosis lung disease. A better understanding of CFTR regulatory mechanisms could suggest new therapeutic strategies. AMP-activated protein kinase (AMPK) binds to and phosphorylates CFTR, attenuating PKA-activated CFTR gating. However, the requirement for AMPK binding to CFTR and the potential role of other proteins in this regulation are unclear. We report that nucleoside diphosphate kinase A (NDPK-A) interacts with both AMPK and CFTR in overlay blots of airway epithelial cell lysates. Binding studies in Xenopus oocytes and transfected HEK-293 cells revealed that a CFTR peptide fragment that binds AMPK (CFTR-1420-57) disrupted the AMPK-CFTR interaction. Introduction of CFTR-1420-57 into human bronchial Calu-3 cells enhanced forskolin-stimulated whole cell conductance in patch clamp measurements. Similarly, injection of CFTR-1420-57 into Xenopus oocytes blocked the inhibition of cAMP-stimulated CFTR conductance by AMPK in two-electrode voltage clamp studies. AMPK also inhibited CFTR conductance with co-expression of WT NDPK-A in two-electrode voltage clamp studies, but co-expression of a catalytically inactive H118F mutant or various Ser-120 NDPK-A mutants prevented this inhibition. In vitro phosphorylation of WT NDPK-A was enhanced by purified active AMPK, but phosphorylation was prevented in H118F and phosphomimic Ser-120 NDPK-A mutants. AMPK does not appear to phosphorylate NDPK-A directly but rather promotes an NDPK-A autophosphorylation event that involves His-118 and Ser-120. Taken together, these results suggest that NDPK-A exists in a functional cellular complex with AMPK and CFTR in airway epithelia, and NDPK-A catalytic function is required for the AMPK-dependent regulation of CFTR.

Chitosan/halloysite Nanotubes Bionanocomposites: Structure, Mechanical Properties and Biocompatibility

Incorporation of nanosized reinforcements into chitosan usually results in improved properties and changed microstructures. Naturally occurred halloysite nanotubes (HNTs) are incorporated into chitosan for forming bionanocomposite films via solution casting. The electrostatic attraction and hydrogen bonding interactions between HNTs and chitosan are confirmed. HNTs are uniformly dispersed in chitosan matrix. The tensile strength and Young's modulus of chitosan are enhanced by HNTs. The storage modulus and glass transition temperature of chitosan/HNTs films also increase significantly. Blending with HNTs induces changes in surface nanotopography and increase of roughness of chitosan films. In vitro fibroblasts response demonstrates that both chitosan and chitosan/HNTs nanocomposite films are cytocompatibility even when the loading of HNTs is 10%. In summary, these results provide insights into understanding of the structural relationships of chitosan/HNTs bionanocomposite films in potential applications, such as scaffold materials in tissue engineering.

ABCA1, ABCG1, and SR-BI: Transit of HDL-associated Sphingosine-1-phosphate

Sphingosine-1-phosphate (S1P) is a zwitterionic lysophospholipid generated by the sphingosine kinase-catalyzed phosphorylation of sphingosine. A number of the biological effects of S1P are mediated by its binding to five specific G protein-coupled receptors located on the cell surface or intracellular targets. However, the synthesis and secretion of S1P require release out of cells for binding with receptors by certain transporters and carriers. High-density lipoprotein (HDL) is an important carrier of S1P in the blood, but the mechanism by which it does so is unclear. This review discusses the mechanism how S1P is transported, and focuses particularly on how the formation of HDL-associated S1P (HDL-S1P) is mediated by certain transporters and carriers. A hypothesis that the ATP-binding cassette transporter A1 (ABCA1), ABCG1, and scavenger receptor class B memberI (SR-BI) play pivotal roles in HDL-S1P formation is also described.

Hydrogen Sulfide Protects HUVECs Against Hydrogen Peroxide Induced Mitochondrial Dysfunction and Oxidative Stress

Hydrogen sulfide (H(2)S) has been shown to have cytoprotective effects in models of hypertension, ischemia/reperfusion and Alzheimer's disease. However, little is known about its effects or mechanisms of action in atherosclerosis. Therefore, in the current study we evaluated the pharmacological effects of H(2)S on antioxidant defenses and mitochondria protection against hydrogen peroxide (H(2)O(2)) induced endothelial cells damage.

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