Bradykinin-induced p42/p44 MAPK phosphorylation and cell proliferation via Src, EGF receptors, and PI3-K/Akt in vascular smooth muscle cells

In our previous study, bradykinin (BK) exerts its mitogenic effect through Ras/Raf/MEK/MAPK pathway in vascular smooth muscle cells (VSMCs). In addition to this pathway, the non-receptor tyrosine kinases (Src), EGF receptor (EGFR), and phosphatidylinositol 3-kinase (PI3-K) have been implicated in linking a variety of G-protein coupled receptors to MAPK cascades. Here, we investigated whether these different mechanisms participating in BK-induced activation of p42/p44 MAPK and cell proliferation in VSMCs. We initially observed that BK- and EGF-dependent activation of Src, EGFR, Akt, and p42/p44 MAPK and [3H]thymidine incorporation were mediated by Src and EGFR, because the Src inhibitor PP1 and EGFR kinase inhibitor AG1478 abrogated BK- and EGF-dependent effects. Inhibition of PI3-K by LY294002 attenuated BK-induced Akt and p42/p44 MAPK phosphorylation and [3H]thymidine incorporation, but had no effect on EGFR phosphorylation, suggesting that EGFR may be an upstream component of PI3-K/Akt and MAPK in these responses. This hypothesis was supported by the tranfection with dominant negative plasmids of p85 and Akt which significantly attenuated BK-induced Akt and p42/p44 MAPK phosphorylation. Pretreatment with U0126 (a MEK1/2 inhibitor) attenuated the p42/p44 MAPK phosphorylation and [3H]thymidine incorporation stimulated by BK, but had no effect on Akt activation. Moreover, BK-induced transactivation of EGFR and cell proliferation was blocked by matrix metalloproteinase inhibitor GM6001. These results suggest that, in VSMCs, the mechanism of BK-stimulated activation of p42/p44 MAPK and cell proliferation was mediated, at least in part, through activation of Src family kinases, EGFR transactivation, and PI3-K/Akt.     

Structural and Biochemical Characterization of Yeast Monothiol Glutaredoxin Grx6

Glutaredoxins (Grxs) are a ubiquitous family of proteins that reduce disulfide bonds in substrate proteins using electrons from reduced glutathione (GSH). The yeast Saccharomyces cerevisiae Grx6 is a monothiol Grx that is localized in the endoplasmic reticulum and Golgi compartments. Grx6 consists of three segments, a putative signal peptide (M1-I36), an N-terminal domain (K37-T110), and a C-terminal Grx domain (K111-N231, designated Grx6C). Compared to the classic dithiol glutaredoxin Grx1, Grx6 has a lower glutathione disulfide reductase activity but a higher glutathione S-transferase activity. In addition, similar to human Grx2, Grx6 binds GSH via an iron-sulfur cluster in vitro. The N-terminal domain is essential for noncovalent dimerization, but not required for either of the above activities. The crystal structure of Grx6C at 1.5 Å resolution revealed a novel two-strand antiparallel β-sheet opposite the GSH binding groove. This extra β-sheet might also exist in yeast Grx7 and in a group of putative Grxs in lower organisms, suggesting that Grx6 might represent the first member of a novel Grx subfamily.     

Analysis and Comparison of Electrical Pulse Parameters for Gene Electrotransfer of Two Different Cell Lines

Knowledge of the parameters which influence the efficiency of gene electrotransfer has importance for practical implementation of electrotransfection for gene therapy as well as for better understanding of the underlying mechanism. The focus of this study was to analyze the differences in gene electrotransfer and membrane electropermeabilization between plated cells and cells in a suspension in two different cell lines (CHO and B16F1). Furthermore, we determined the viability and critical induced transmembrane voltage (ITV_c) for both cell lines. In plated cells we obtained relatively little difference in electropermeabilization and gene electrotransfection between CHO and B16F1 cells. However, significant differences between the two cell lines were observed in a suspension. CHO cells exhibited a much higher gene electrotransfection rate compared to B16F1 cells, whereas B16F1 cells reached maximum electropermeabilization at lower electric fields than CHO cells. Both in a suspension and on plated cells, CHO cells had a slightly better survival rate at higher electric fields than B16F1 cells. Calculation of ITV_c in a suspension showed that, for both electropermeabilization and gene electrotransfection, CHO cells have lower ITV_c than B16F1 cells. In all cases, ITV_c for electropermeabilization was lower than ITV_c for gene electrotransfer, which is in agreement with other studies. Our results show that there is a marked difference in the efficiency of gene electrotransfer between suspended and plated cells.     

Location of the analgesic domain in Scorpion toxin BmK AGAP by mutagenesis of disulfide bridges

An increasing number of analgesic peptides have been found in the tail toxicyst, but there has been little research into their analgesic domains. Where are the analgesic domains in a conservative βαββ topology conformation of the analgesic peptides? We have carried out research to address this question. On account of the importance of disulfide bonds in the study of protein structure, the conformational stability, catalytic activity and folding, and site-directed mutagenesis in disulfide bridges have been used to look for the analgesic domain in a mature antitumor-analgesic peptide from the venom of the Chinese scorpion Buthus martensii Karsch (BmK AGAP). The mouse-twisting assay was used to examine the analgesic activity of 12 mutants, in which two mutants (C22S, C46S) and (C16S, C36S), exhibited lower relative activity. Following the conformational analysis, one domain, called the “core domain”, was found to be the key to the analgesic activity.     

Measuring Brain Uptake and Incorporation into Brain Phosphatidylinositol of Plasma myo-[2H6]Inositol in Unanesthetized Rats: An Approach to Estimate In vivo Brain Phosphatidylinositol Turnover

The in vivo rate of turnover of phosphatidylinositol (PtdIns) in brain is not known. In brain, certain receptor-mediated signal transduction involves metabolism of PtdIns and a method to measure its turnover in awake animals is useful in studying the effect of lithium and other therapeutic agents. In a method described here, rats were infused subcutaneously with myo-[²H₆]inositol (Ins*) using an osmotic pump and, at 1 and 8 weeks, concentrations of free myo-inositol (Ins) and Ins* in plasma and brain were measured by GC-MS (chemical ionization). Also, PtdIns and PtdIns* together in brain were isolated, and Ins and Ins* from their headgroups were released enzymatically and specific activity of incorporated inositol was measured. The specific activity of inositol reached a steady state in plasma within 1 week of infusion, but not in brain even at 8 weeks. However, in brain, the specific activity of phosphatidylinositol was same as that of inositol at both time-points, suggestive of fast turnover of PtdIns. The animal experiment and the analytical methodology described here should be useful for measuring the rate of turnover of brain PtdIns in pathological and drug treatment conditions.     

Expression of Discoidin Domain Receptor 2 (DDR2) Extracellular Domain in Pichia Pastoris and Functional Analysis in Synovial Fibroblasts and NIT3T3 Cells

Discoidin domain receptor 2 (DDR2) is a kind of protein tyrosine kinases associated with cell proliferation and tumor metastasis, and collagen, identified as a ligand for DDR2, up-regulates matrix metallloproteinase 1 (MMP-1) and MMP-2 expression in cellular matrix. To investigate the roles of DDR2 in destruction of cartilage in rheumatoid arthritis (RA) and tumor metastasis, we tried to express extracellular domain of DDR2 fused with a His tag to increase protein solubility and facilitate purification (without signal peptide and transmembrane domain, designated DR) in Pichia pastoris, purify the expressed protein, and characterize its function, for purpose of future application as a specific DDR2 antagonist. Two clones of relative high expression of His-DR were obtained, After purification by a Ni-NTA (nitric-tri-acetic acid) chromatographic column, soluble fused His-DR over 90% purity were obtained. Competitive binding inhibition assay demonstrated that expressed His-DR could block the binding of DDR2 and natural DDR2 receptors on NIT3T3 and synovial cell surfaces. Results of RT-PCR, Western blotting, and gelatinase zymography showed that His-DR was capable of inhibiting MMP-1 and MMP-2 secretion from NIT3T3 cells and RA synoviocytes stimulated by collagen II. For MMP-1, the inhibitory effect was displayed at the levels of mRNA and protein, whereas for MMP-2 it was demonstrated at the level of protein physiological activity. All these findings suggested that the fused expressed His-DR inhibited the activity of natural DDR2, and relevant MMP-1 and MMP-2 expression in synoviocytes and NIH3T3 cells provoked by collagen II. Wei Zhang and Tianbing Ding equally contributed to this work.     

Influence of chk1 and plk1 silencing on radiation- or cisplatin-induced cytotoxicity in human malignant cells

The G2/M checkpoint is an attractive pathway for targeting and sensitizing tumor cells to cancer treatment. Abrogation of the G2/M checkpoint by targeting molecules, such as checkpoint kinase 1 (chk1), increases DNA breakage and sensitizes tumor cells to anti-tumoral agents. However, most of the previously described G2/M abrogators are actually targeting the G2-M border checkpoints rather than mitotic checkpoints. This prompted us to test the effects of combined targeting of chk1 and a critical regulator of mitosis, polo-like kinase 1 (plk1). Chk1 and plk1 were found to be co-expressed in 70% of primary neoplastic tissues we examined. Asynchronized tumor cells were treated with different DNA damaging-agents to activate G1/S, S or G2/M checkpoints. Either chk1 or plk1-specific antisense oligodeoxynucleotides (ASODN) enhanced DNA damaging agent-induced apoptosis. When used in combination, however, chk1- plus plk1-specific ASODN failed to produce synergistic effects. Moreover, selective targeting of plk1 or chk1 in tumor xenografts of mice by oncolytic adenovirus mutants demonstrated potent anti-tumoral efficacy in the presence of low dose cisplatin. Again, combined targeting of chk1 and plk1 did not further enhance anti-tumoral efficacy. We concluded that combined targeting of chk1 and plk1 was not superior to either targeting chk1 or plk1 alone, which suggested that chk1 and plk1 silencing might overlap in their mechanism of action. Whether combined targeting of chk1 with other, more specific mitotic regulators would synergistically sensitize tumor to anti-neoplastic therapeutics needs to be further clarified. Qinglei Gao and Xiaoyuan Huang contributed equally to this work.