A p-Tyr42 RhoA Inhibitor Promotes the Regeneration of Human Corneal Endothelial Cells by Ameliorating Cellular Senescence.

The development of treatment strategies for human corneal endothelial cells (hCECs) disease is necessary because hCECs do not regenerate in vivo due to the properties that are similar to senescence. This study is performed to investigate the role of a p-Tyr42 RhoA inhibitor (MH4, ELMED Inc., Chuncheon) in transforming growth factor-beta (TGF-ß)- or H2O2-induced cellular senescence of hCECs. Cultured hCECs were treated with MH4. The cell shape, proliferation rate, and cell cycle phases were analyzed. Moreover, cell adhesion assays and immunofluorescence staining for F-actin, Ki-67, and E-cadherin were performed. Additionally, the cells were treated with TGF-ß or H2O2 to induce senescence, and mitochondrial oxidative reactive oxygen species (ROS) levels, mitochondrial membrane potential, and NF-κB translocation were evaluated. LC3II/LC3I levels were determined using Western blotting to analyze autophagy. MH4 promotes hCEC proliferation, shifts the cell cycle, attenuates actin distribution, and increases E-cadherin expression. TGF-ß and H2O2 induce senescence by increasing mitochondrial ROS levels and NF-κB translocation into the nucleus; however, this effect is attenuated by MH4. Moreover, TGF-ß and H2O2 decrease the mitochondrial membrane potential and induce autophagy, while MH4 reverses these effects. In conclusion, MH4, a p-Tyr42 RhoA inhibitor, promotes the regeneration of hCECs and protects hCECs against TGF-ß- and H2O2-induced senescence via the ROS/NF-κB/mitochondrial pathway.

Discovery of GCC5694A: A potent and selective sodium glucose co-transporter 2 inhibitor for the treatment of type 2 diabetes.

Sodium-dependent glucose co-transporter 2 (SGLT2) has emerged as a promising drug target for the treatment of type 2 diabetes, and recently, several SGLT2 inhibitors have been approved for clinical use. A series of molecules with a C-aryl glucoside scaffold was designed and synthesized for biological evaluation. Among the molecules tested, a dihydrobenzofuran-containing analog, 14g (GCC5694A), exhibited excellentin vitro activity against SGLT2 (IC50 = 0.460 nM), good selectivity for SGLT1, and good metabolic stability. Data from further evaluation of the compound in animal models showed that this molecule is a promising candidate for development as an anti-diabetic agent.

Task specificity impacts dual-task interference in older adults.

BACKGROUND: Task prioritization is an important factor determines the magnitude and direction of dual-task interference in older adults. Greater dual-task cost during walking may lead to falling, sometimes causing lasting effects on mobility. AIMS: We investigated dual-task interference for walking and cognitive performance. METHODS: Twenty healthy, older adults (71 ± 5 years) completed three cognitive tasks: letter fluency, category fluency, and serial subtraction during seated and walking conditions on a self-paced treadmill for 3 min each, in addition to walking only condition. Walking speed, step length and width were measured during walking and each dual-task condition. RESULTS: Comparing the percentage of correct answers in cognitive tasks across single and dual-task conditions, there was a main effect of cognitive task (p = 0.021), showing higher scores during letter fluency compared to serial subtraction (p = 0.011). Step width was significantly wider during dual-task letter fluency compared to walking alone (p = 0.003), category fluency (p = 0.001), and serial subtraction (p = 0.007). DISCUSSION: During both fluency tasks, there was a cost for gait and cognition, with category showing a slightly higher cognitive cost compared to letter fluency. During letter fluency, to maintain cognitive performance, gait was sacrificed by increasing step width. During serial subtraction, there was a cost for gait, yet a benefit for cognitive performance. CONCLUSION: Differential effect of cognitive task on dual-task performance is critical to be understood in designing future research or interventions to improve dual-task performance of most activities of daily living.

Antioxidant and Anti-Inflammatory Activities in Relation to the Flavonoids Composition of Pepper (Capsicum annuum L.).

The chili pepper (Capsicum annuum L.) is a food source that is rich in flavonoids such as luteolin and apigenin. Flavonoids are known to have anti-inflammatory and antioxidant activities; however, studies on the flavonoids composition identified and the anti-inflammatory and antioxidant effects in pepper leaves (PL) and fruits (PF) are insufficient. In the present study, we investigated the antioxidant and anti-inflammatory effects in vitro, and the flavonoids contents of the PL and PF. Pepper extracts showed radical scavenging activities and ameliorated the lipopolysaccharide (LPS)-stimulated inflammatory response by decreasing nitric oxide production and interluekin-6 and tumor necrosis factor alpha levels in RAW 264.7 cells, with more effective activities noted for PL than for PF. Furthermore, PL extracts markedly inhibited the LPS-induced production of reactive oxygen species accumulation. The flavonoid profile and content of pepper were dependent on the part, with PL showing higher total flavonoids than PF. In particular, the content of luteolin glycosides in PL was twice that in PF. Thus, PL may be useful to prevent oxidative stress and inflammation-related diseases.

Establishment of Effective Mouse Model of Premature Ovarian Failure Considering Treatment Duration of Anticancer Drugs and Natural Recovery Time.

OBJECTIVES: This study was aimed to establish the most effective premature ovarian failure (POF) mouse model using Cyclophosphamide (CTX), busulfan (Bu), and cisplatin considering treatment duration of anticancer drugs and natural recovery time. METHODS: POF was induced by intraperitoneally injecting CTX (120 mg/kg)/Bu (12 mg/kg) for 1 to 4 weeks or cisplatin (2 mg/kg) for 3 to 14 days to C57BL/6 female mice aged 6 to 8 weeks. Controls were injected with equal volume of saline for the same periods. Body weight was measured every week, and ovarian and uterine weights were measured after the last injection of anticancer drug. To assess ovarian function, POF-induced mice were superovulated with pregnant mare serum gonadotropin and human chorionic gonadotropin, and then mated with male. After 18 hours, zygotes were retrieved and cultured for 4 days. Finally, the mice were left untreated for a period of times after the final injection of anticancer drug, and the time for natural recovery of ovarian function was evaluated. RESULTS: After 2 weeks of CTX/Bu injection, ovarian and uterine weights, and ovarian function were decreased sharply. Cisplatin treatment for 10 days resulted in a significant decrease in ovarian and uterine weight, and ovarian function. When POF was induced for at least 2 weeks for CTX/Bu and for at least 10 days for cisplatin, ovarian function did not recover naturally for 2 weeks and 1 week, respectively. CONCLUSIONS: These results suggest that CTX/Bu should be treated for at least 2 weeks and cisplatin for at least 10 days to establish the most effective primary ovarian insufficiency mouse model.

Pap1p-dependent upregulation of thioredoxin 3 and thioredoxin reductase genes from the fission yeast under nitrosative stress.

The thioredoxin system, consisting of thioredoxin, thioredoxin reductase, and NADPH, is involved in the response against a variety of stresses. The TRX3(+) and TrxR(+) genes encode thioredoxin 3 and thioredoxin reductase, respectively, in the fission yeast Schizosaccharomyces pombe . Their transcriptional regulations were studied using the lacZ fusion genes. Synthesis of ß-galactosidase from the TRX3(+)-lacZ fusion gene was markedly enhanced by nitric-oxide-generating sodium nitroprusside in the Pap1p-positive cells but not in the Pap1p-negative cells. Similarly, synthesis of ß-galactosidase from the TrxR(+)-lacZ fusion gene was upregulated by sodium nitroprusside in a Pap1p-dependent manner. Synthesis of ß-galactosidase from the TRX3(+)-lacZ and TrxR(+)-lacZ fusion genes was also enhanced by S-nitrosoglutathione in the Pap1p-positive cells but not in the Pap1p-negative cells. In brief, the S. pombe genes encoding thioredoxin 3 and thioredoxin reductase are upregulated under nitrosative stress in a Pap1p-dependent manner.

The extracellular loop 2 of TM4SF5 inhibits integrin alpha2 on hepatocytes under collagen type I environment.

Four-transmembrane L6 family member 5 (TM4SF5) and its homolog L6, a tumor antigen, form a four-transmembrane L6 family. TM4SF5 expression causes uncontrolled cell proliferation and angiogenesis. Although other genuine transmembrane 4 superfamily (TM4SF) members co-operate with integrins for cell migration, roles of TM4SF5 in the cellular spreading and migration are unknown. Using hepatocarcinoma cell clones that ectopically express TM4SF5, we found that cross talks via an extracellular interaction between TM4SF5 and integrin alpha2 in collagen type I environment inhibited integrin alpha2 functions such as spreading on and migration toward collagen I, which were recovered by suppression of TM4SF5 or structural disturbance of its second extracellular loop using a peptide or mutagenesis. Altogether, the observations suggest that TM4SF5 in hepatocytes negatively regulates integrin alpha2 function via an interaction between the extracellular loop 2 of TM4SF5 and integrin alpha2 during cell spreading on and migration through collagen I environment.

Blockade of four-transmembrane L6 family member 5 (TM4SF5)-mediated tumorigenicity in hepatocytes by a synthetic chalcone derivative.

UNLABELLED: We previously reported that the four-transmembrane L6 family member 5 (TM4SF5) was highly expressed in hepatocarcinoma, induced morphological elongation and epithelial-mesenchymal transition, and caused abnormal cell growth in multilayers in vitro and tumor formation in vivo. In this study, we identified a synthetic compound, 4'-(p-toluenesulfonylamido)-4-hydroxychalcone (TSAHC) that antagonized both the TM4SF5-mediated multilayer growth and TM4SF5-enhanced migration/invasion. TSAHC treatment induced multilayer-growing cells to grow in monolayers, recovering contact inhibition without accompanying apoptosis, and inhibited chemotactic migration and invasion. Tumor formation in nude mice injected with TM4SF5-expressing cells and the growth of cells expressing endogenous TM4SF5, but not of TM4SF5-null cells, was suppressed by treatment with TSAHC, but not by treatment with its analogs. The structure-activity relationship indicated the significance of 4'-p-toluenesulfonylamido and 4-hydroxy groups for the anti-TM4SF5 effects of TSAHC. Point mutations of the putative N-glycosylation sites abolished the TM4SF5-specific TSAHC responsiveness. CONCLUSION: These observations suggest that TM4SF5-enhanced tumorigenic proliferation and metastatic potential can be blocked by TSAHC, likely through targeting the extracellular region of TM4SF5, which is important for protein-protein interactions.

Cooperation between integrin alpha5 and tetraspan TM4SF5 regulates VEGF-mediated angiogenic activity.

Tetraspan TM4SF5 is highly expressed in a diverse number of tumor types. Here we explore the mechanistic roles of TM4SF5 in angiogenesis. We found that TM4SF5 overexpression correlates with vascular endothelial growth factor (VEGF) expression in SNU449 hepatocytes and with vessel formation in clinical hepatocarcinoma samples. Conditioned media from TM4SF5-expressing cells enhanced viability and tube formation of primary human umbilical vein endothelial cells, and outgrowth of endothelial cells from aorta ring segments, which was abolished by treatment with an anti-VEGF antibody. TM4SF5 retained integrin alpha(5) on the cell surface for VEGF induction, and preincubation with anti-integrin alpha(5) antibody abolished TM4SF5-mediated VEGF expression and secretion. TM4SF5-mediated effects required integrin alpha(5), c-Src, and signal transducer and activator of transcription 3 (STAT3). In addition, tumors from nude mice injected with TM4SF5-expressing cells and from clinical human hepatocarcinoma tissues showed enhanced integrin alpha(5) expression, vessel formation, and signaling activity, which were inhibited by administration of anti-integrin alpha(5) or -VEGF antibody. This study suggests that TM4SF5 facilitates angiogenesis of neighboring endothelial cells through VEGF induction, mediated by cooperation between TM4SF5 and integrin alpha(5) of epithelial cells.

The pap1(+) gene of fission yeast is transcriptionally regulated by nitrosative and nutritional stress.

In the current work, regulation of the pap1(+) gene was investigated by the use of the pap1(+)-lacZ fusion gene and semi-quantitative reverse transcriptase-PCR. The synthesis of beta-galactosidase from the pap1(+)-lacZ fusion gene was significantly enhanced by nitric oxide (NO)-generating sodium nitroprusside (SNP) and nitrogen starvation. However, the induction by SNP and nitrogen starvation was observed to be much less in the Pap1p-negative cells harboring the fusion gene. Exogenous NO was more effectively scavenged in the Pap1p-positive cells than in the Pap1p-negative cells. Oxidative stress such as superoxide anion, hydrogen peroxide and cadmium could not give rise to an effect on the synthesis of beta-galactosidase from the fusion gene. The pap1(+) mRNA level was elevated in the wild-type cells by SNP and nitrogen starvation. Catalase activity, a major enzyme positively regulated by Pap1p, was significantly increased only in the Pap1p-positive cells by SNP. In brief, it is demonstrated that transcription of the Schizosaccharomyces pombe pap1(+) gene is positively regulated by nitrosative and nutritional stress in a Pap1p-dependent manner.