Effect of doxycycline-regulated calnexin and calreticulin expression on specific thrombopoietin productivity of recombinant Chinese hamster ovary cells

In an attempt to increase the specific thrombopoietin (TPO) productivity (q(TPO)) of recombinant Chinese hamster ovary (rCHO) cells (CHO-TPO), the effect of expression level of calnexin (CNX) and calreticulin (CRT) on q(TPO) was investigated. To control both CNX and CRT expression levels simultaneously, the Tet-Off system was first introduced in CHO-TPO cells, and stable Tet-Off cells (TPO-Tet-Off) were screened by luciferase assay. The doxycycline-regulated CNX and CRT expression system in rCHO cells (TPO-CNX/CRT) was established by cotransfection of CNX and CRT expression vector and pTK-Hyg vector into TPO-Tet-Off cells and subsequent screening by Western blot analysis of CNX and CRT. The expression levels of CNX and CRT in TPO-CNX/CRT cells could be tightly controlled by adding different concentrations of doxycycline to a culture medium. Compared with the basal level (2 microg/mL doxycyline), a 2.9-fold increase in CNX expression and a 2.8-fold increase in CRT expression were obtained in the absence of doxycycline. This, in turn, resulted in a 1.9-fold increase in q(TPO), not inhibiting cell growth or changing in vivo biological activity of TPO. Taken together, these results demonstrate that a simultaneous overexpression of CNX and CRT can increase the q(TPO) of rCHO cells.     

Anti-apoptotic effect of insulin-like growth factor (IGF)-I and its receptor in porcine preimplantation embryos derived from in vitro fertilization and somatic cell nuclear transfer

Insulin-like growth factor (IGF)-I is a receptor-mediated autocrine and/or paracrine growth and/or survival factor for mammalian embryo development. It is known to promote the growth and development of mouse preimplantation embryos. The present study was designed to investigate the effects of IGF-I (50 ng/ml), anti-IGF-I receptor antibody (50 ng/ml) and their combination on porcine preimplantation embryo development. Furthermore, the mechanism underlying the embryotropic effects of IGF-I was evaluated by monitoring the incidence of apoptosis and expression of apoptosis-related genes. In both in vitro fertilized (IVF) and somatic cell nuclear transfer (SCNT) embryos, culturing with IGF-I increased the rate of blastocyst formation and this embryotrophic effect was neutralized by culturing with IGF-I along with anti-IGF-I receptor (IGF-IR) antibody. Culturing IVF and SCNT embryos with IGF-I significantly increased the number of total cells in blastocysts and decreased the number of apoptotic nuclei. These effects of IGF-I were also neutralized by culturing with IGF-I along with anti-IGF-IR antibody. Expression of the anti-apoptotic Bcl-2 gene was increased, while expression of the pro-apoptotic Bax was decreased in both IVF and SCNT embryos cultured with IGF-I. In both IVF and SCNT embryos, anti-IGF-IR antibody along with IGF-I neutralized the effect of IGF-I on expression of Bcl-2 and Bax genes. In conclusion, the present study demonstrated that IGF-I through its specific receptors improved the developmental competence of IVF and SCNT embryos by decreasing the incidence of apoptosis and regulating apoptosis-related genes in porcine preimplantation embryos.     

Column-switching high-performance liquid chromatographic method for the determination of zaltoprofen in rat plasma

A direct injection column-switching high-performance liquid chromatography (HPLC) method was developed and validated for quantification of zaltoprofen in rat plasma. Following dilution with mobile phase A, i.e. acetonitrile-10mM potassium phosphate buffer (pH 6.8) (12:88, v/v) samples were directly injected to the pre-column without sample pre-purification step. After endogenous plasma components were eluted to waste, the system was switched and the analyte was eluted to the trap column. Zaltoprofen was then back-flushed to the analytical column for separation with mobile phase B, i.e. acetonitrile-10mM potassium phosphate buffer (pH 6.8) (35:65, v/v) and quantification with an ultraviolet detector at 230 nm. The calibration curve was linear in the concentration range of 40-5000 ngmL(-1). This method has been fully validated and shown to be specific, accurate and precise. The method is simple, rapid and the sample preparation is minimal and appears to be useful for the pharmacokinetic study of zaltoprofen.     

Surface plasmon resonance imaging protein arrays for analysis of triple protein interactions of HPV, E6, E6AP, and p53

We have developed a surface plasmon resonance (SPR)-based protein microarray to study protein-protein interactions in a high-throughput mode. As a model system, triple protein interactions have been explored with human papillomaviral E6 protein, tumor suppressor p53, and ubiquitin ligase E6AP. Human papillomavirus (HPV) is known to be a causative agent of cervical cancer. Upon infection, the viral E6 protein forms a heterotrimeric protein complex with p53 and E6AP. The formation of the complex eventually results in the degradation of p53. In the present study, a GST-fused E6AP protein was layered onto a glutathione (GSH)-modified gold chip surface. The specific binding of GST-E6AP protein onto the gold chip surface was facilitated through the affinity of GST to its specific ligand GSH. The interacting proteins (E6 and/or p53) were then spotted. Detection of the interaction was performed using a SPR imaging (SPRI) technique. The resulting SPRI intensity data showed that the protein-protein interactions of E6AP, E6, and p53 were detected in a concentration-dependent manner, suggesting that the SPRI-based microarray system can be an effective tool to study protein-protein interactions where multiple proteins are involved.     

Comparison of Newly Generated Doublecortin-immunoreactive Neuronal Progenitors in the Main Olfactory Bulb among Variously Aged Gerbils

In the present study, we investigated age-related differences in neuronal progenitors in the gerbil main olfactory bulb (MOB) using doublecortin (DCX), a marker for neuronal progenitors which differentiate into neurons in the brain. No difference in the number of neuronal nuclei (NeuN)-immunoreactive neurons was found in the MOB at variously aged gerbils. At postnatal month (PM) 1, DCX immunoreaction was detected in all layers of the MOB except for the olfactory nerve layer. At this time point, DCX-immunoreactive cells (neuronal progenitors) were very abundant; however, they did not have fully developed-processes. From PM 3, the number of DCX-immunoreactive neuronal progenitors was decreased with age. At PM 6, DCX-immunoreactive cells showed very well-developed processes. In western blot analysis, DCX protein level in the MOB was highest at PM 1. Thereafter, levels of DCX protein were decreased with age. In the subventricular zone of the lateral ventricle, the number of Ki-67-immunoractive cells (proliferating cells) was also significantly decreased with age. In addition, increases of α-synuclein-immunoreactive structures were observed in the MOB with age. These results suggest that decrease in DCX-immunoreactive neuronal progenitors and its protein levels in the MOB with age may be associated with reduction of cell proliferation in the SVZ and with an increase in α-synuclein in the MOB.     

Design,synthesis, and binding of homologated truncated 4′-thioadenosine derivatives at the human A3 adenosine receptors

We synthesized homologated truncated 4′-thioadenosine analogues 3 in which a methylene (CH₂) group was inserted in place of the glycosidic bond of a potent and selective A₃ adenosine receptor antagonist 2. The analogues were designed to induce maximum binding interaction in the binding site of the A₃ adenosine receptor. However, all homologated nucleosides were devoid of binding affinity at all subtypes of adenosine receptors, indicating that free rotation through the single bond allowed the compound to adopt an indefinite number of conformations, disrupting the favorable binding interaction essential for receptor recognition.     

Cytokine GM-CSF genetic adjuvant facilitates prophylactic DNA vaccine against pseudorabies virus through enhanced immune responses

Granulocyte/macrophage colony-stimulatory factor (GM-CSF) is an attractive adjuvant for a DNA vaccine on account of its ability to recruit antigen-presenting cells (APCs) to the site of antigen synthesis as well as its ability to stimulate the maturation of dendritic cells (DCs). This study evaluated the utility of GM-CSF cDNA as a DNA vaccine adjuvant for glycoprotein B (gB) of pseudorabies virus (PrV) in a murine model. The co-injection of GM-CSF DNA enhanced the levels of serum PrV-specific IgG with a 1.5-to 2-fold increase. Moreover, GM-CSF co-injection inhibited the production of IgG2a isotype. However, it enhanced production of an IgG1 isotype resulting in humoral responses biased to the Th2-type against PrV antigen. In contrast, the co-administration of GM-CSF DNA enhanced the T cell-mediated immunity biased to the Th1-type, as judged by the significantly higher level of cytokine IL-2 and IFN-gamma production but not IL-4. When challenged with a lethal dose of PrV, the GM-CSF co-injection enhanced the resistance against a PrV infection. This suggests that co-inoculation with a vector expressing GM-CSF enhanced the protective immunity against a PrV infection. This immunity was caused by the induction of increased humoral and cellular immunity in response to PrV antigen.