Expression and Changes of Hyperoxidized Peroxiredoxins in Non-Pyramidal and Polymorphic Cells in the Gerbil Hippocampus During Normal Aging

Oxidative stress is one of predisposing factors to age-related neurodegeneration in the brain. In particular, thiol-containing groups are susceptible to oxidative stress, which induces the formation of the disulfide bond and/or hyperoxidized form of thiol-containing proteins. We observed the protein thiol levels in the hippocampal homogenates and also investigated changes in hyperoxidized form of peroxiredoxin (Prx–SO₃) immunoreactivity and proteins levels in the gerbil hippocampal subregions during normal aging. Levels of total thiol, non-protein thiol, and protein thiol were decreased in the hippocampal homogenates with age. At post-natal month 1 (PM 1), pyramidal and non-pyramidal cells in the hippocampal CA1 region (CA1) showed Prx–SO₃ immunoreactivity. Prx–SO₃ immunoreactivity in the cells was decreased by PM 12, thereafter, Prx–SO₃ immunoreactivity in the cells increased again with age. In the CA2/3, Prx–SO₃ immunoreactivity in pyramidal cells was not significantly changed; however, the immunoreactivity in pyramidal cells was very low at PM 12. Prx–SO₃ immunoreactivity in the dentate gyrus (DG) was distinctly changed during aging. At PM 1, Prx–SO₃ immunoreactivity in granule and polymorphic cells was weak and strong, respectively. The immunoreactivity in the neurons was decreased with age, not shown in any neurons at PM 12. Thereafter, Prx–SO₃ immunoreactivity increased again with age. In addition, Prx–SO₃ protein level in the hippocampus was lowest at PM 12. These results suggest that thiol-containing proteins are changed during aging and Prx–SO₃ immunoreactivity was different according to cells in the hippocampal subregion during aging.     

(2-Aryl-5-methylimidazol-4-ylcarbonyl)guanidines and (2-aryl-5-methyloxazol-4-ylcarbonyl)guanidines as NHE-1 inhibitors

A series of (2-aryl-5-methylimidazol-4-ylcarbonyl)guanidines and (2-aryl-5-methyloxazol-4-ylcarbonyl)guanidines were synthesized and evaluated as NHE-1 inhibitors. The structure–activity relationships well matched those of furan derivatives, which were previously investigated. The (2,5-disubstituted)phenyl compounds showed better activities than the other analogues in both imidazole and oxazole compounds. Especially, 2-(2,5-dichlorophenyl)imidazole 52, and 2-(2-methoxy-5-chlorophenyl)imidazole 54 compounds exhibited potent cardioprotective efficacy both in vitro and in vivo as well as high NHE-1 inhibitory activities.     

Design,synthesis, screening,and molecular modeling study of a new series of ROS1 receptor tyrosine kinase inhibitors

A series of rationally designed ROS1 tyrosine kinase inhibitors was synthesized and screened. Compound 12b has showed good potency with IC₅₀ value of 209 nM, which is comparable with that of the reference lead compound 1. Molecular modeling studies have been performed, that is, a homology model for ROS1 was built, and the screened inhibitors were docked into its major identified binding site. The docked poses along with the activity data have revealed a group of the essential features for activity. Overall, simplification of the lead compound 1 into compound 12b has maintained the activity, while facilitated the synthetic advantages. A molecular interaction model for ROS1 kinase and inhibitors has been proposed.     

Phosphate-buffered saline-based nucleofection of primary endothelial cells

Although various nonviral transfection methods are available, cell toxicity, low transfection efficiency, and high cost remain hurdles for in vitro gene delivery in cultured primary endothelial cells. Recently, unprecedented transfection efficiency for primary endothelial cells has been achieved due to the newly developed nucleofection technology that uses a combination of novel electroporation condition and specific buffer components that stabilize the cells in the electrical field. Despite superior transfection efficiency and cell viability, high cost of the technology has discouraged cardiovascular researchers from liberally adopting this new technology. Here we report that a phosphate-buffered saline (PBS)-based nucleofection method can be used for efficient gene delivery into primary endothelial cells and other types of cells. Comparative analyses of transfection efficiency and cell viability for primary arterial, venous, microvascular, and lymphatic endothelial cells were performed using PBS. Compared with the commercial buffers, PBS can support equally remarkable nucleofection efficiency to both primary and nonprimary cells. Moreover, PBS-mediated nucleofection of small interfering RNA (siRNA) showed more than 90% knockdown of the expression of target genes in primary endothelial cells. We demonstrate that PBS can be an unprecedented economical alternative to the high-cost buffers or nucleofection of various primary and nonprimary cells.     

Apoptosis signal-regulating kinase 1 regulates the expression of caspase-11

Caspase-11 is an inducible caspase involved in the regulation of cell death and inflammation. In the present study, we examined whether apoptosis signal-regulating kinase 1 (Ask1)-mediated signaling pathway is involved in the expression of caspase-11 induced by lipopolysaccharide (LPS). We found that the induction of caspase-11 was suppressed by the inhibitors of NADPH oxidase (Nox) or knockdown of Nox4 that acts downstream of toll-like receptor 4 and generates Ask1-activating reactive oxygen species. Overexpression of dominant negative tumor necrosis factor receptor associate factor 6 also suppressed the induction of caspase-11. Importantly, knockdown or dominant negative form of Ask1 suppressed the induction of caspase-11 following LPS stimulation. Taken together, our results show that Ask1 regulates the expression of caspase-11 following LPS stimulation.     

Differential expression of cell surface proteins in human bone marrow mesenchymal stem cells cultured with or without basic fibroblast growth factor containing medium

Mesenchymal stem cells (MSCs) are multipotent cells, which have the capability to differentiate into various mesenchymal tissues such as bone, cartilage, fat, tendon, muscle, and marrow stroma. However, they lose the capability of multi‐lineage differentiation after several passages. It is known that basic fibroblast growth factor (bFGF) increases growth rate, differentiation potential, and morphological changes of MSCs in vitro. In this report, we have used 2‐DE coupled to MS to identify differentially expressed proteins at the cell membrane level in MSCs growing in bFGF containing medium. The cell surface proteins isolated by the biotin–avidin affinity column were separated by 2‐DE in triplicate experiments. A total of 15 differentially expressed proteins were identified by quadrupole‐time of flight tandem MS. Nine of the proteins were upregulated and six proteins were downregulated in the MSCs cultured with bFGF containing medium. The expression level of three actin‐related proteins, F‐actin‐capping protein subunit alpha‐1, actin‐related protein 2/3 complex subunit 2, and myosin regulatory light chain 2, was confirmed by Western blot analysis. The results indicate that the expression levels of F‐actin‐capping protein subunit alpha‐1, actin‐related protein 2/3 complex subunit 2, and myosin regulatory light chain 2 are important in bFGF‐induced morphological change of MSCs.     

Verification of Beam Models for Ionic Polymer-Metal Composite Actuator

Ionic Polymer-Metal Composite (IPMC) can work as an actuator by applying a few voltages.A thick IPMC actuator,whereNafion-117 membrane was synthesized with polypyrrole/alumina composite filler,was analyzed to verify the equivalent beamand equivalent bimorph beam models.The blocking force and tip displacement of the IPMC actuator were measured with a DCpower supply and Young’s modulus of the IPMC strip was measured by bending and tensile tests respectively.The calculatedmaximum tip displacement and the Young’s modulus by the equivalent beam model were almost identical to the correspondingmeasured data.Finite element analysis with thermal analogy technique was utilized in the equivalent bimorph beam model tonumerically reproduce the force-displacement relationship of the IPMC actuator.The results by the equivalent bimorph beammodel agreed well with the force-displacement relationship acquired by the measured data.It is confirmed that the equivalentbeam and equivalent bimorph beam models are practically and effectively suitable for predicting the tip displacement,blockingforce and Young’s modulus of IPMC actuators with different thickness and different composite of ionic polymer membrane.     

Phylogenetic diversification of glycogen synthase kinase 3/SHAGGY-like kinase genes in plants

Background  

The glycogen synthase kinase 3 (GSK3)/SHAGGY-like kinases (GSKs) are non-receptor serine/threonine protein kinases that are involved in a variety of biological processes. In contrast to the two members of the GSK3 family in mammals, plants appear to have a much larger set of divergent GSK genes. Plant GSKs are encoded by a multigene family; analysis of the Arabidopsis genome revealed the existence of 10 GSK genes that fall into four major groups. Here we characterized the structure of Arabidopsis and rice GSK genes and conducted the first broad phylogenetic analysis of the plant GSK gene family, covering a taxonomically diverse array of algal and land plant sequences.