Characterization of the DNA binding activity of structural protein VP1 from chicken anaemia virus.

BACKGROUND: Chicken anaemia virus (CAV) is commonly found in poultry. VP1 is the sole structural protein of CAV, which is the major component responsible for capsid assembly. The CAV virion consists of the VP1 protein and a viral genome. However, there is currently no information on the protein-nucleic acid interactions between VP1 and DNA molecules. RESULTS: In this study, the recombinant VP1 protein of CAV was expressed and purified to characterize its DNA binding activity. When VP1 protein was incubated with a DNA molecule, the DNA molecule exhibited retarded migration on an agarose gel. Regardless of whether the sequence of the viral genome was involved in the DNA molecule, DNA retardation was not significantly influenced. This outcome indicated VP1 is a DNA binding protein with no sequence specificity. Various DNA molecules with different conformations, such as circular dsDNA, linear dsDNA, linear ssDNA and circular ssDNA, interacted with VP1 proteins according to the results of a DNA retardation assay. Further quantification of the amount of VP1 protein required for DNA binding, the circular ssDNA demonstrated a high affinity for the VP1 protein. The preferences arranged in the order of affinity for the VP1 protein with DNA are circular ssDNA, linear ssDNA, supercoiled circular dsDNA, open circular DNA and linear dsDNA. CONCLUSIONS: The results of this study demonstrated that the interaction between VP1 and DNA molecules exhibited various binding preferences that were dependent on the structural conformation of DNA. Taken together, the results of this report are the first to demonstrate that VP1 has no sequence-specific DNA binding activity. The particular binding preferences of VP1 might play multiple roles in DNA replication or encapsidation during the viral life cycle.

Ultrasonography and clinical outcome comparison of extracorporeal shock wave therapy and corticosteroid injections for chronic plantar fasciitis: A randomized controlled trial.

OBJECTIVES: Extracorporeal shockwave therapy (ESWT) and corticosteroid injection (CSI) are treatment options for plantar fasciitis. Their clinical outcome comparison remains a debate. Also, the thickness changes of the plantar fascia on objective evaluation under the medium energy ESWT and CSI therapy are elusive. METHODS: A total of 97 patients with chronic plantar fasciitis were enrolled in the randomized prospective trial. Forty-seven patients received extracorporeal shock wave therapy (ESWT), and fifty patients received corticosteroid injection (CSI). The thickness of the plantar fascia was evaluated respectively before ESWT and CSI, and at the 4th and 12th week after ESWT and CSI by ultrasonography. Pain level and clinical outcomes were recorded using visual analogue scale (VAS) and 100-points scoring systems. Correlation analysis was performed between the thickness change and clinical outcome. RESULTS: Under ultrasonography, we observed more increase of plantar fascia thickness of ESWT group than CSI group at 4th week (p=0.048). VAS of plantar fasciitis patients receiving ESWT was lower than those who received corticosteroid injection (0.001 and p⟨0.001, at 4th and 12th week). On the assessment of 100-points scoring systems, the pain level of patients with ESWT was lower than those with CSI at the 12th week (p⟨0.001). On the other hand, the increase of plantar fascia thickness at 4th week was positively correlated with the decrease of VAS score at 12th week follow-up (R=0.302, P=0.039). CONCLUSIONS: At 4th week after treatment, the thickness of plantar fascia increased. Then it decreased gradually, but not to the baseline at 12th week. On the pain level outcome at 12th week, extracorporeal shockwave therapy (ESWT) was more efficient than corticosteroid injection (CSI) on chronic plantar fasciitis. The more change of plantar fascia after ESWT, the more efficient on clinical outcome.

VP2 of Chicken Anaemia Virus Interacts with Apoptin for Down-regulation of Apoptosis through De-phosphorylated Threonine 108 on Apoptin.

Chicken anaemia virus (CAV) is an important contagious agent that causes immunosuppressive disease in chickens. CAV Apoptin is a nucleoplasmic shuffling protein that induces apoptosis in chicken lymphoblastoid cells. In the present study, confocal microscopy revealed co-localisation of expressed CAV non-structural protein VP2 with Apoptin in the nucleus of MDCC-MSB1 cells and the nucleoplasmic compartment of CHO-K1 cells. In vitro pull-down and ex vivo biomolecular fluorescent complementation (BiFC) assays further showed that the VP2 protein directly interacts with Apoptin. Transient co-expression of VP2 and Apoptin in MDCC-MSB1 cells significantly decreased the rate of apoptosis compared with that in cells transfected with the Apoptin gene alone. In addition, the phosphorylation status of threonine 108 (Thr108) of Apoptin was found to decrease upon interaction with VP2. Although dephosphorylated Thr108 did not alter the subcellular distribution of Apoptin in the nucleus of MDCC-MSB1 cells, it did suppress apoptosis. These findings provide the first evidence that VP2 directly interacts with Apoptin in the nucleus to down-regulate apoptosis through alterations in the phosphorylation status of the latter. This information will be useful to further elucidate the underlying mechanism of viral replication in the CAV life cycle.

Rapid and sensitive identification of the herbal tea ingredient Taraxacum formosanum using loop-mediated isothermal amplification.

Taraxacum formosanum (TF) is a medicinal plant used as an important component of health drinks in Taiwan. In this study, a rapid, sensitive and specific loop-mediated isothermal amplification (LAMP) assay for authenticating TF was established. A set of four specific LAMP primers was designed based on the nucleotide sequence of the internal transcribed spacer 2 (ITS2) nuclear ribosomal DNA (nrDNA) of TF. LAMP amplicons were successfully amplified and detected when purified genomic DNA of TF was added in the LAMP reaction under isothermal condition (65 °C) within 45 min. These specific LAMP primers have high specificity and can accurately discriminate Taraxacum formosanum from other adulterant plants; 1 pg of genomic DNA was determined to be the detection limit of the LAMP assay. In conclusion, using this novel approach, TF and its misused plant samples obtained from herbal tea markets were easily identified and discriminated by LAMP assay for quality control.

Involvement of calcium-mediated reactive oxygen species in inductive GRP78 expression by geldanamycin in 9L rat brain tumor cells.

Treatment with geldanamycin (GA) leads to an increase in [Ca2+]c and the production of reactive oxygen species (ROS) in rat brain tumor 9L RBT cells. GA-exerted calcium signaling was blocked by BAPTA/AM and EGTA. The effect of GA on [Ca2+]c was significantly reduced in the presence of thapsigargin (TG) and ruthenium red (RR). GA-induced GRP78 expression is significantly decreased in the presence of BAPTA/AM, EGTA and RR, suggesting that the calcium influx from the extracellular space and intracellular calcium store oscillations are contributed to by the calcium mobilization and GRP78 expression induced by GA. The induced GRP78 expression is sensitive to added U73122 and Ro-31-8425, pinpointing the involvement of phospholipase C (PLC) and protein kinase C (PKC) in GA-induced endoplasmic reticulum (ER) stress. The antioxidants N-acetylcysteine (NAC), BAPTA/AM, EGTA and H7 also have significant inhibitory effects on ROS generation. Finally, neither H7 nor NAC was able to affect the calcium response elicited by GA. Our results suggest that the causal signaling cascade during GA-inducted GRP78 expression occurs via a pathway that connects PLC to cytoplasmic calcium increase, PKC activation and, then, finally, ROS generation. Our data provides new insights into the influence of GA on ER stress response in 9L RBT cells.

Efficient production of an engineered apoptin from chicken anemia virus in a recombinant E. coli for tumor therapeutic applications.

BACKGROUND: Apoptin, a nonstructural protein encoded by the VP3 gene of chicken anemia virus (CAV), has been shown to not only induce apoptosis when introduced into the precursors of chicken thymocytes, but has been found to specifically kill human cancer cells, tumor cell and transformed cells without affecting the proliferation of normal cells. This tumor-specific apoptotic characteristic of the protein potentially may allow the development of a protein drug that has applications in tumor therapy. However, several major problems, which include poor expression and poor protein solubility, have hampered the production of apoptin in bacteria. RESULTS: Significantly increased expression of recombinant full-length apoptin that originated from chicken anemia virus was demonstrated using an E. coli expression system. The CAV VP3 gene was fused with a synthetic sequence containing a trans-acting activator of transcription (TAT) protein transduction domain (PTD). The resulting construct was cloned into various different expression vectors and these were then expressed in various E. coli strains. The expression of the TAT-Apoptin in E. coli was significantly increased when TAT-Apoptin was fused with GST-tag rather than a His-tag. When the various rare amino acid codons of apoptin were optimized, the expression level of the GST-TAT-Apoptin(opt) in E. coli BL21(DE3) was significantly further increased. The highest protein expression level obtained was 8.33 g/L per liter of bacterial culture after induction with 0.1 mM IPTG for 4 h at 25 °C. Moreover, approximately 90% of the expressed GST-TAT-Apoptin(opt) under these conditions was soluble. After purification by GST affinity chromatography, the purified recombinant TAT-Apoptin(opt) protein was used to evaluate the recombinant protein's apoptotic activity on tumor cells. The results demonstrated that the E. coli-expressed GST-TAT-apoptin(opt) showed apoptotic activity and was able to induce human premyelocytic leukemia HL-60 cells to enter apoptosis. CONCLUSIONS: On expression in E. coli, purified recombinant TAT-Apoptin(opt) that has been fused to a GST tag and had its codons optimized, was found to have great potential. This protein may in the future allow the development of a therapeutic protein that is able to specifically kill tumor cells.

Development and characterization of a potential diagnostic monoclonal antibody against capsid protein VP1 of the chicken anemia virus.

Chicken anemia virus (CAV) is an important viral pathogen that causes anemia and severe immunodeficiency syndrome in chickens worldwide. In this study, a potential diagnostic monoclonal antibody against the CAV VP1 protein was developed which can precisely recognize the CAV antigen for diagnostic and virus recovery purposes. The VP1 gene of CAV encoding the N-terminus-deleted VP1 protein, VP1Nd129, was cloned into an Escherichia (E.) coli expression vector. After isopropyl-ß-D-thiogalactopyronoside induction, VP1Nd129 protein was shown to be successfully expressed in the E. coli. By performing an enzyme-linked immunoabsorbent assay using two coating antigens, purified VP1Nd129 and CAV-infected liver tissue lysate, E3 monoclonal antibody (mAb) was found to have higher reactivity against VP1 protein than the other positive clones according to the result of limiting dilution method from 64 clones. Using immunohistochemistry, the presence of the VP1-specific mAb, E3, was confirmed using CAV-infected liver and thymus tissues as positive-infected samples. Additionally, CAV particle purification was also performed using an immunoaffinity column containing E3 mAb. The monoclonal E3 mAb developed in this study will not only be very useful for detecting CAV infection and performing histopathology studies of infected chickens, but may also be used to purify CAV particles in the future.

Identification of the NLS and NES motifs of VP2 from chicken anemia virus and the interaction of VP2 with mini-chromosome maintenance protein 3.

BACKGROUND: VP2 of chicken anemia virus (CAV) is a dual-specificity phosphatase required for virus infection, assembly and replication. The functions of the nuclear localization signal (NLS) and nuclear export signal (NES) of VP2 in the cell, however, are poorly understood. Our study identified the presence of a NLS in VP2 and showed that the protein interacted significantly with mini-chromosome maintenance protein 3 (MCM3) in the cell. RESULTS: An arginine-lysine rich NLS could be predicted by software and spanned from amino acids 133 to 138 of VP2. The critical amino acids residues between positions 136 and 138, and either residue 133 or 134 are important for nuclear import in mammalian cells based on systematic mutagenesis. A NES is also predicted in VP2; however the results suggest that no functional NES is present and that this protein is CRM1 independent. It was also shown that VP2 is a chromatin binding protein and, notably, using a co-immunoprecipitation assay, it was found that VP2 association with MCM3 and that this interaction does not require DSP activity. CONCLUSIONS: VP2 contains a NLS that span from amino acids 133 to 138. VP2 is a CRM1 independent protein during nuclear export and associates with MCM3 in cells.

High yield expression in a recombinant E. coli of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development.

BACKGROUND: Chicken anemia virus (CAV), the causative agent chicken anemia, is the only member of the genus Gyrovirus of the Circoviridae family. CAV is an immune suppressive virus and causes anemia, lymph organ atrophy and immunodeficiency. The production and biochemical characterization of VP1 protein and its use in a subunit vaccine or as part of a diagnostic kit would be useful to CAV infection prevention. RESULTS: Significantly increased expression of the recombinant full-length VP1 capsid protein from chicken anemia virus was demonstrated using an E. coli expression system. The VP1 gene was cloned into various different expression vectors and then these were expressed in a number of different E. coli strains. The expression of CAV VP1 in E. coli was significantly increased when VP1 was fused with GST protein rather than a His-tag. By optimizing the various rare amino acid codons within the N-terminus of the VP1 protein, the expression level of the VP1 protein in E. coli BL21(DE3)-pLysS was further increased significantly. The highest protein expression level obtained was 17.5 g/L per liter of bacterial culture after induction with 0.1 mM IPTG for 2 h. After purification by GST affinity chromatography, the purified full-length VP1 protein produced in this way was demonstrated to have good antigenicity and was able to be recognized by CAV-positive chicken serum in an ELISA assay. CONCLUSIONS: Purified recombinant VP1 protein with the gene's codons optimized in the N-terminal region has potential as chimeric protein that, when expressed in E. coli, may be useful in the future for the development of subunit vaccines and diagnostic tests.

The suitable restriction enzymes for pulsed-field gel electrophoresis analysis of Bordetella pertussis.

We searched the restriction enzymes with rare cutting sites on the genome of Bordetella pertussis strain Tohama I using the Restriction Digest Tool software provided in the Institute for Genomic Research web site. The usefulness of 5 enzymes for pulsed-field gel electrophoresis (PFGE) analysis was evaluated with 68 B. pertussis isolates. The results indicated that AflII, DraI, SpeI, and XbaI were useful enzymes, and AflII was the best one for PFGE analysis of B. pertussis isolates.

Associations of the major pseudopilin XpsG with XpsN (GspC) and secretin XpsD of Xanthomonas campestris pv. campestris type II secretion apparatus revealed by cross-linking analysis.

The major pseudopilin XpsG is an essential component of type II secretion apparatus of Xanthomonas campestris pv. campestris. Along with other ancillary pseudopilins, it forms a pilus-like structure spanning between cytoplasmic and outer membranes. Associations of pseudopilins with non-pseudopilin members of type II secretion apparatus were not well documented, probably due to their dynamic or unstable nature. In this study, by treating intact cells with a cleavable cross-linker dithiobis(succinimidylpropionate) (DSP), followed by metal chelating chromatography and immunoblotting on secretion-positive strains of X. campestris pv. campestris, we discovered associations of XpsGh with XpsN (GspC), as well as XpsD. These associations were detectable in a strain missing all components, but XpsO, of the type II secretion apparatus. However, chromosomal non-polar mutation in each gene exerted different effects upon the association between the other two. The XpsGh/XpsD association is undetectable in xpsN mutant; however, it was restored to a limited extent by overproducing XpsD protein. The XpsGh/XpsN association is unaltered by a lack of XpsD protein or an elevation of its abundance. Co-immune precipitation between XpsN and XpsD, while being independent of XpsG, was nonetheless enhanced by raising XpsG protein level. These observations agree with the proposition that the type II secretion apparatus in a cell may exist as an integrated multiprotein complex with all components working in concert. Moreover, in functional machinery, the association of the major pseudopilin XpsG with secretin XpsD appears strongly dependent on the existence of XpsN, the GspC protein.

XpsG, the major pseudopilin in Xanthomonas campestris pv. campestris, forms a pilus-like structure between cytoplasmic and outer membranes.

GspG, -H, -I, -J and -K proteins are members of the pseudopilin family. They are the components required for the type II secretion pathway, which translocates proteins across the outer membrane of Gram-negative bacteria to the extracellular milieu. They were predicted to form a pilus-like structure, and this has been shown for PulG of Klebsiella oxytoca by using electron microscopy. In the present study, we performed biochemical analyses of the XpsG protein of Xanthomonas campestris pv. campestris and observed that it is a pillar-like structure spanning the cytoplasmic and outer membranes. Subcellular fractionation revealed a soluble form (SF) of XpsG, in addition to the membrane form. Chromatographic analysis of SF XpsG in the absence of a detergent indicated that it is part of a large complex (>440 kDa). In vitro studies indicated that XpsG is prone to aggregate in the absence of a detergent. We isolated and characterized a non-functional mutant defective in forming the large complex. It did not interfere with the function of wild-type XpsG and was not detectable in the SF. Moreover, unlike wild-type XpsG, which was distributed in both the cytoplasmic and outer membranes, it appeared only in the cytoplasmic membrane. When wild-type XpsG was co-expressed with His6-tagged XpsH but not with untagged XpsH, SF XpsG bound to nickel and co-eluted with XpsH. This result suggests the presence of other pseudopilin components in the XpsG-containing large-sized molecules.