Rapid Antibacterial Activity Assessment of Chimeric Lysins.

Various chimeric lysins have been developed as efficacious antibiotics against multidrug-resistant bacteria, but direct comparisons of their antibacterial activities have been difficult due to the preparation of multiple recombinant chimeric lysins. Previously, we reported an Escherichia coli cell-free expression method to better screen chimeric lysins against Staphylococcus aureus, but we still needed to increase the amounts of expressed proteins enough to be able to detect them non-isotopically for quantity comparisons. In this study, we improved the previous cell-free expression system by adding a previously reported artificial T7 terminator and reversing the different nucleotides between the T7 promoter and start codon to those of the T7 phage. The new method increased the expressed amount of chimeric lysins enough for us to detect them using Western blotting. Therefore, the qualitative comparison of activity between different chimeric lysins has become possible via the adjustment of the number of variables between samples without protein purification. We applied this method to select more active chimeric lysins derived from our previously reported chimeric lysin (ALS2). Finally, we compared the antibacterial activities of our selected chimeric lysins with reported chimeric lysins (ClyC and ClyO) and lysostaphin and determined the rank orders of antibacterial activities on different Staphylococcus aureus strains in our experimental conditions.

Kv3 channels contribute to cancer cell migration via vimentin regulation.

Cell migration is a complex and important process in cancer progression. Vimentin has pivotal roles in cancer cell migration, and various signaling pathways including the AKT pathway are involved in cancer cell migration via vimentin regulation. Recent studies have revealed that voltage-gated potassium (Kv) channels have important functions in cancer cell migration; however, the exact mechanism is still unclear. In the present study, we focused on Kv3 channels with vimentin in cancer migration using human cervical cancer cells (HeLa) and canine mammary tumor cells (CHMp). Cancer cell migration was significantly inhibited, and vimentin expression was significantly decreased by Kv3 blocker, BDS-II. The Kv3 blocker also inactivated the AKT pathway in HeLa cells. In addition, reduced expressions of vimentin and Kv3.4 were observed in HeLa cells when treated with AKT blocker, MK2206. These results suggest that Kv3 channels play important roles in cancer cell migration by regulating vimentin and having closely related with the AKT pathway in human cervical cancer cells.

Pharmacological inhibition of Kv3 on oxidative stress-induced cataract progression.

Oxidative stress is one of the most important risk factors for cataractogenesis. Previous studies have indicated that BDS-II, a Kv3 channel blocker, plays pivotal roles in oxidative stress-related diseases. This study demonstrates that BDS-II exerts a protective effect on cataractogenesis. Specifically, BDS-II was observed to inhibit lens opacity induced by H2O2. BDS-II was also determined to inhibit cataract progression in a sodium selenite-induced in vivo cataract model by inhibiting reduction of the total GSH. In addition, BDS-II was demonstrated to protect human lens epithelial cells against H2O2-induced cell death. Our results suggest that BDS-II is a potential pharmacological candidate in cataract therapy.

Kv3.4 regulates cell migration and invasion through TGF-ß-induced epithelial-mesenchymal transition in A549 cells.

Epithelial-mesenchymal transition (EMT) is the process by which epithelial cells acquire mesenchymal characteristics. This process induces cell migration and invasion, which are closely related to cancer metastasis and malignancy. EMT consists of various intermediate states that express both epithelial and mesenchymal traits, called partial EMT. Recently, several studies have focused on the roles of voltage-gated potassium (Kv) channels associated with EMT in cancer cell migration and invasion. In this study, we demonstrate the relationship between Kv3.4 and EMT and confirm the effects of cell migration and invasion. With TGF-ß treatment, EMT was induced and Kv3.4 was also increased in A549 cells, human lung carcinoma cells. The knockdown of Kv3.4 blocked the EMT progression reducing cell migration and invasion. However, the Kv3.4 overexpressed cells acquired mesenchymal characteristics and increased cell migration and invasion. The overexpression of Kv3.4 also has a synergistic effect with TGF-ß in promoting cell migration. Therefore, we conclude that Kv3.4 regulates cancer migration and invasion through TGF-ß-induced EMT and these results provide insights into the understanding of cancer metastasis.

The role of the voltage-gated potassium channel, Kv2.1 in prostate cancer cell migration.

Voltage-gated potassium (Kv) channels are involved in many important cellular functions and play pivotal roles in cancer progression. The expression level of Kv2.1 was observed to be higher in the highly metastatic prostate cancer cells (PC-3), specifically in their membrane, than in immortalized prostate cells (WPMY-1 cells) and comparatively less metastatic prostate cancer cells (LNCaP and DU145 cells). However, Kv2.1 expression was significantly decreased when the cells were treated with antioxidants, such as N-acetylcysteine or ascorbic acid, implying that the highly expressed Kv2.1 could detect reactive oxygen species (ROS) in malignant prostate cancer cells. In addition, the blockade of Kv2.1 with stromatoxin-1 or siRNA targeting Kv2.1 significantly inhibited the migration of malignant prostate cancer cells. Our results suggested that Kv2.1 plays an important role as a ROS sensor and that it is a promising therapeutic molecular target in metastasis of prostate cancer. [BMB Reports 2021; 54(2): 130-135].

The Presence and Distribution of TRPM7 in the Canine Mammary Glands.

The transient receptor potential melastatin-subfamily member 7 (TRPM7) cation channel is a bifunctional ion channel with intrinsic kinase activity and is ubiquitously expressed in the animal/human body. Accumulated knowledge of TRPM7 suggests that it plays an essential role in normal physiological processes, including the development, survival, proliferation, differentiation, and migration of cells. The aim of this study was to demonstrate the presence and expression patterns of TRPM7 in normal canine mammary glands using reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemistry. Normal mammary gland tissue samples were obtained from five female beagle dogs. RT-PCR and sequencing of the amplified PCR products demonstrated the presence of TRPM7 mRNA in normal mammary glands, and the presence of TRPM7 protein was confirmed by Western blotting. Immunohistochemical investigations demonstrated the expression of TRPM7 in the apical membrane of acinar and ductal epithelial cells in the canine mammary glands. These results provide the first evidence of the presence and distribution of TRPM7 in the canine mammary gland and could help explain the physiological and pathological roles of TRPM7 in the canine mammary gland; however, additional studies are required to elucidate these roles.