Enhanced Mutant Screening in One-step PCR-based Multiple Site-directed Plasmid Mutagenesis by Introduction of Silent Restriction Sites for Structural and Functional Study of Proteins.

Site-directed mutagenesis (SDM) has been widely used for studying the structure and function of proteins. A one-step polymerase chain reaction (PCR)-based multiple site-directed plasmid mutagenesis method with extended non-overlapping sequence at the 3' end of the primer increases the PCR amplification efficiency and the capacity of multi-site mutagenesis. Here, we introduced silent restriction sites in the primers used in this PCR-based SDM method by utilizing SDM-Assist software to generate mutants of Helicobacter pylori neutrophil-activating protein (HP-NAP), whose gene has low GC content. The HP-NAP mutants were efficiently generated by this modified mutagenesis method and quickly identified by a simple restriction digest due to the presence of the silent restriction site. This modified PCR-based SDM method with the introduction of a silent restriction site on the primer is efficient for generation and identification of mutations in the gene of interest.

Progesterone modulates cell growth via integrin αvß3-dependent pathway in progesterone receptor-negative MDA-MB-231 cells.

Progesterone (P4) plays a pivotal role in regulating the cancer progression of various types, including breast cancer, primarily through its interaction with the P4 receptor (PR). In PR-negative breast cancer cells, P4 appears to function in mediating cancer progression, such as cell growth. However, the mechanisms underlying the roles of P4 in PR-negative breast cancer cells remain incompletely understood. This study aimed to investigate the effects of P4 on cell proliferation, gene expression, and signal transduction in PR-negative MDA-MB-231 breast cancer cells. P4-activated genes, associated with proliferation in breast cancer cells, exhibit a stimulating effect on cell growth in PR-negative MDA-MB-231 cells, while demonstrating an inhibitory impact in PR-positive MCF-7 cells. The use of arginine-glycine-aspartate (RGD) peptide successfully blocked P4-induced extracellular signal-regulated kinase 1/2 (ERK1/2) activation, aligning with computational models of P4 binding to integrin αvß3. Disrupting integrin αvß3 binding with RGD peptide or anti-integrin αvß3 antibody altered P4-induced expression of proliferative genes and modified P4-induced cell growth in breast cancer cells. In conclusion, integrin αvß3 appears to mediate P4-induced ERK1/2 signal pathway to regulate proliferation via alteration of proliferation-related gene expression in PR-negative breast cancer cells.

Glucose biosensors based on a gold nanodendrite modified screen-printed electrode.

In this study, an enzymatic glucose biosensor based on a three-dimensional gold nanodendrite (GND) modified screen-printed electrode was developed. The GNDs were electrochemically synthesized on the working electrode component of a commercially available screen-printed electrode using a solution acquired by dissolving bulk gold in aqua regia as the precursor. The 3D GND electrode greatly enhanced the effective sensing area of the biosensor, which improved the sensitivity of glucose detection. Actual glucose detections demonstrated that the fabricated devices could perform at a sensitivity of 46.76 µA mM⁻¹ cm⁻² with a linear detection range from 28 µM-8.4 mM and detection limit of 7 µM. A fast response time (∼3 s) was also observed. Moreover, only a 20 µl glucose oxidase is required for detection owing to the incorporation of the commercially available screen-printed electrode.

Integrins and Actions of Androgen in Breast Cancer.

Androgen has been shown to regulate male physiological activities and cancer proliferation. It is used to antagonize estrogen-induced proliferative effects in breast cancer cells. However, evidence indicates that androgen can stimulate cancer cell growth in estrogen receptor (ER)-positive and ER-negative breast cancer cells via different types of receptors and different mechanisms. Androgen-induced cancer growth and metastasis link with different types of integrins. Integrin αvß3 is predominantly expressed and activated in cancer cells and rapidly dividing endothelial cells. Programmed death-ligand 1 (PD-L1) also plays a vital role in cancer growth. The part of integrins in action with androgen in cancer cells is not fully mechanically understood. To clarify the interactions between androgen and integrin αvß3, we carried out molecular modeling to explain the potential interactions of androgen with integrin αvß3. The androgen-regulated mechanisms on PD-L1 and its effects were also addressed.

Protease-activated receptor 2 induces migration and promotes Slug-mediated epithelial-mesenchymal transition in lung adenocarcinoma cells.

Protease-activated receptor 2 (PAR2), a G protein-coupled receptor for trypsin, contributes to growth, anti-apoptosis, and migration in lung cancer. Given that PAR2 activation in airway epithelial cells compromises the airway epithelium barrier by disruption of E-cadherin adhesion, PAR2 may be involved in epithelial-mesenchymal transition (EMT) in lung adenocarcinoma cells. Although PAR2 is known to promote the migration of lung cancer cells, the detailed mechanism of this event is still not clear. Here, we found that PAR2 is highly expressed in several lung adenocarcinoma cell lines. In two lung adenocarcinoma cell lines, CL1-5 and H1299 cells, activation of PAR2 induces migration and Slug-mediated EMT. The underlying mechanisms involved in PAR2-induced migration and EMT in CL1-5 cells were further investigated. We showed that PAR2-induced migration of CL1-5 cells is mediated by the Src/p38 mitogen-activated protein kinase (p38 MAPK) signaling pathway. ß-arrestin 1, not G protein, is involved in this PAR2-mediated Src/p38 MAPK signaling pathway. PAR2-induced EMT in CL1-5 cells is dependent on the activation of extracellular-signal-regulated kinase 2 (ERK2). The activation of ERK2 further mediates Slug stabilization through suppressing the activity of glycogen synthase kinase 3ß. In addition, a poor prognosis was observed in lung adenocarcinoma patients with a high expression of PAR2. Thus, PAR2 regulates migration through ß-arrestin 1-dependent activation of p38 MAPK and EMT through ERK2-mediated stabilization of Slug in lung adenocarcinoma cells. Our finding also suggests that PAR2 might serve as a therapeutic target for metastatic lung adenocarcinoma and a potential biomarker for predicting the prognosis of lung adenocarcinoma.

Endocytosis-dependent lysosomal degradation of Src induced by protease-activated receptor 1.

Src plays a critical role in regulating cellular responses induced by protease-activated receptor 1 (PAR1). Here, we found that PAR1 activation induces lysosomal degradation of Src. Src is associated and trafficked together with activated PAR1 to early endosomes and then sorted to lysosomes for degradation. Blocking agonist-induced endocytosis of PAR1 by inhibition of dynamin activity suppresses PAR1-induced degradation of Src. However, Src activity is neither required for agonist-induced PAR1 internalization nor required for Src degradation upon PAR1 activation. We show that PAR1 activation triggers endocytosis-dependent lysosomal degradation of Src in both human embryonic kidney 293 and human umbilical vein endothelial cells. Our finding provides a new paradigm for how an irreversibly activated receptor regulates its downstream signalling.

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode.

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. pylori-induced gastric inflammation by activating several innate leukocytes including neutrophils, monocytes, and mast cells. The immunogenic and immunomodulatory properties of HP-NAP make it a potential diagnostic and vaccine candidate for H. pylori and a new drug candidate for cancer therapy. In order to obtain substantial quantities of purified HP-NAP used for its clinical applications, an efficient method to purify this protein with high yield and purity needs to be established. In this protocol, we have described a method for one-step negative chromatographic purification of recombinant HP-NAP overexpressed in Escherichia coli (E. coli) by using diethylaminoethyl (DEAE) ion-exchange resins (e.g., Sephadex) in batch mode. Recombinant HP-NAP constitutes nearly 70% of the total protein in E. coli and is almost fully recovered in the soluble fraction upon cell lysis at pH 9.0. Under the optimal condition at pH 8.0, the majority of HP-NAP is recovered in the unbound fraction while the endogenous proteins from E. coli are efficiently removed by the resin. This purification method using negative mode batch chromatography with DEAE ion-exchange resins yields functional HP-NAP from E. coli in its native form with high yield and purity. The purified HP-NAP could be further utilized for the prevention, treatment, and prognosis of H. pylori-associated diseases as well as cancer therapy.

Helicobacter pylori neutrophil-activating protein induces release of histamine and interleukin-6 through G protein-mediated MAPKs and PI3K/Akt pathways in HMC-1 cells.

Helicobacter pylori neutrophil-activating protein (HP-NAP) activates several innate leukocytes including neutrophils, monocytes, and mast cells. It has been reported that HP-NAP induces degranulation and interleukin-6 (IL-6) secretion of rat peritoneal mast cells. However, the molecular mechanism is not very clear. Here, we show that HP-NAP activates human mast cell line-1 (HMC-1) cells to secrete histamine and IL-6. The secretion depends on pertussis toxin (PTX)-sensitive heterotrimeric G proteins but not on Toll-like receptor 2. Moreover, HP-NAP induces PTX-sensitive G protein-mediated activation of extracellular signal-regulated kinase 1/2 (ERK1/2), p38-mitogen-activated protein kinase (p38 MAPK), and Akt in HMC-1 cells. Inhibition of ERK1/2, p38 MAPK, or phosphatidylinositol 3-kinase (PI3K) suppresses HP-NAP-induced release of histamine and IL-6 from HMC-1 cells. Thus, the activation of HMC-1 cells by HP-NAP is through Gi-linked G protein-coupled receptor-mediated MAPKs and PI3K/Akt pathways.