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Rapid eye movement sleep (REMS) plays an important role in maintaining brain excitability by regulating noradrenaline (NA) level and Na-K ATPase activity. We showed earlier that REMS deprivation (REMSD) associated elevated NA increased neuronal, while decreased glial Na-K ATPase activity. However, our knowledge was insufficient on how the REMSD-associated effect is sustained particularly under chronic condition. Using Neuro-2a cells as a model, we investigated the molecular mechanism of NA-induced increase in mRNA expression of Na-K ATPase subunit and the enzyme activity. The cells were treated with NA in the presence or absence of either α1- or β-adrenoceptor (AR) antagonists, Ca ++ -channel blocker or SERCA-inhibitor, and PKA or PKC inhibitor. We observed that NA acting on α1-AR increased Na-K ATPase activity and mRNA expression of the catalytic α1- and α3-Na-K ATPase subunits in the Neuro-2a cells. Further, PLC and PKC mediated modulation of intracellular Ca ++ played a critical role in inducing the mRNA expression. On the other hand NA, acting on β-AR up-regulated expression of the regulatory β1-subunit of Na-K ATPase. The involvement of SP1 as well as phospho-CREB transcription factors in the NA-mediated increased expression of various subunit isoforms was established. The results of this study along with that of earlier reports support our proposed working model of NA-induced increase in mRNA expression of specific Na-K ATPase subunit leading to increased Na-K ATPase activity. The findings help us understand the molecular mechanism of NA-induced increased brain excitability, for example, upon REMSD including under chronic condition., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Objective: To investigate hydrogen sulfide and its synthases, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), in human prostatic tissue and cells., Methods: CBS and CSE in human prostatic tissue and cells were located using immunostaining. Western blot and a sulfur-sensitive electrode were used to evaluate the expression levels and catalytic activity of CBS and CSE. We analyzed the association between dihydrotestosterone-added or hormone-reduced medium-induced CBS/CSE protein levels with androgen receptor levels in prostate cancer lines. All experiments were repeated ≥3 times., Results: Endogenous hydrogen sulfide and its synthases existed in various areas of human prostatic tissue and cells. Cell activity and CBS/CSE protein levels were greatest in the androgen-dependent prostate cancer cell LNCaP among all cells and downregulated by dihydrotestosterone., Conclusion: Hydrogen sulfide and its synthases in human prostatic tissue and cells were modulated by dihydrotestosterone, which could suggest a potential therapy for prostatic disease., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Chitosan derivatives such as N,N,N-trimethylated chitosan (TMC) are currently being investigated for the delivery of drugs, vaccines and genes. However, the influence of the extent of N-acetylation of these polymers on their enzymatic degradability and biological properties is unknown. In this study, TMCs with a degree of acetylation (DA) ranging from 11 to 55% were synthesized by using a three-step method. First, chitosan was partially re-acetylated using acetic anhydride followed by quantitative dimethylation using formaldehyde and sodium borohydrate. Then, in presence of an excess amount of iodomethane, TMC was synthesized. The TMCs obtained by this method showed neither detectable O-methylation nor loss in acetyl groups ((1)H NMR) and a slight increase in molecular weight (GPC) with increasing degree of substitution, implying that no chain scission occurred during synthesis. The extent of lysozyme-catalyzed degradation of TMC, and that of its precursors chitosan and dimethyl chitosan, was highly dependent on the DA and polymers with the highest DA showed the largest decrease in molecular weight. On Caco-2 cells, TMCs with a high DA ( approximately 50%), a DQ of around 44% and with or without O-methylated groups, were not able to open tight junctions in the trans-epithelial electrical resistance (TEER) assay, in contrast with TMCs (both O-methylated and O-methyl free; concentration 2.5mg/ml) with a similar DQ but a lower DA which were able to reduce the TEER with 30 and 70%, respectively. Additionally, TMCs with a high DA ( approximately 50%) demonstrated no cell toxicity (MTT, LDH release) up to a concentration of 10mg/ml.

Epidemiological and experimental animal data indicate that exposure to both metals and metalloid species exacerbates the risk of human diseases, particularly cancers. Vascular endothelial growth factor (VEGF), which performs a primary function in both tumor progression and angiogenesis, is up-regulated due to exposure to an array of carcinogenic metals, but the mechanisms responsible for the metal activation remain somewhat poorly understood. Recently, we demonstrated that AMP-activated protein kinase (AMPK), which acts as an energy sensor, providing metabolic adaptation effects under ATP-deprived conditions, is critical for the expression of VEGF under oxygen- and glucose-deprived conditions. As carcinogenic metals are potent VEGF expression inducers, we hypothesized that AMPK would also play a crucial role in metal-induced VEGF expression. Here, we present evidence that carcinogenic metals such as arsenite, vanadate, and cobalt, induce AMPK activation and VEGF expression via several different mechanisms, and that AMPK is able to regulate the expression of VEGF mRNA in a hypoxia-inducible factor-1-dependent or -independent manner, depending on the metal applied. We also attempted to characterize the relevant signal transduction pathways in metal-induced VEGF expression and AMPK activation, as well as the role of reactive oxygen species within this context. Overall, our data suggest that AMPK is a critical regulatory component in metal-induced VEGF expression, which further implies its intrinsic involvement in metal-induced carcinogenesis.

The biocompatibility of zirconia-alumina (ZA) nano-composites in load-bearing applications such as dental/orthopedic implants was significantly enhanced by the addition of bioactive HA. The ZA matrix was composed of nano-composite powder obtained from the Pechini process and had higher flexural strength than conventionally mixed zirconia-alumina composite. Because the ZA nano-composite powder effectively decreased the contact area between HA and zirconia for their reaction during the sintering process, the HA-added ZA nano-composites contained biphasic calcium phosphates (BCP) of HA/TCP and had higher flexural strength than conventionally mixed ZA-HA composite. From the in vitro test with osteoblastic cell-lines, the proliferation and the differentiation (as expressed by the alkaline phosphatase activity) of the cellular response on the HA-added ZA nano-composites gradually increased as the amount of HA added increased. From the mechanical and biological evaluations of the HA-added ZA nano-composites, 30HA (30 vol% HA + 70 vol% ZA) was found to be the optimal composition for load-bearing biological applications.

Human NRH:quinone oxidoreductase 2 (NQO2) gene-containing 29-bp deletion/insertion polymorphic promoters were found to be associated with susceptibility to Parkinson's disease. Here, we demonstrate that the NQO2 gene is differentially expressed by the polymorphic promoters in human fibroblasts and Hep-G2 cells transfected with NQO2 gene reporter constructs. Promoter containing the 29-bp insertion polymorphism demonstrated significantly lower NQO2 gene expression. Deletion mutagenesis and DNase I footprinting analysis of the promoter without the 29-bp insertion identified three protected regions (region A, B, and C). Band- and supershift and transfection assays showed binding of transcription factor Sp1 to regions A and B, which regulated expression of the NQO2 gene. Similar studies of the NQO2 gene promoter with the 29-bp insertion polymorphism showed that regions A and C were identical and contributed similarly as in the promoter without the 29-bp insertion to NQO2 gene expression. However, region B was found to be inserted with 29-bp DNA element and bound to both Sp1 and Sp3. Binding of Sp3 led to repression of NQO2 gene transcription by the promoter containing the 29-bp insertion polymorphism. These studies also suggest that alterations in NQO2 activity might be an important factor in susceptibility to Parkinson's disease.

The aim of this study was to investigate the cytotoxic activity of the third-generation nitrogen-containing bisphosphonate zoledronic acid (ZOL) as a single agent, and in combination with clinically relevant anticancer drugs, in a panel of human osteogenic sarcoma cell lines (HOS, BTK-143, MG-63, SJSA-1, G-292, and SAOS2). We found that ZOL, when used alone, reduced cell number in a dose- and time-dependent manner, due either to cell cycle arrest in S-phase or to the induction of apoptosis. In the sensitive HOS, BTK-143, and G-292 cell lines, genomic DNA fragmentation and morphological changes characteristic of apoptosis were evident, and cells became nonadherent. Induction of apoptosis in osteosarcoma cells by ZOL was associated with caspase activation. However, coaddition of the broad-spectrum caspase inhibitors, z-VAD-fmk, Boc-D-fmk, or the caspase-3-specific inhibitor z-DEVD fmk, failed to protect these cells from ZOL-induced apoptosis. Our data support a ZOL-specific induction of cell apoptosis that involves cell detachment (anoikis), and in which caspase activation occurs secondarily to, and is redundant as a mediator of cell death. The addition of geranylgeraniol, an intermediate of the mevalonate pathway, suppressed the ZOL-induced apoptosis, suggesting that the cytotoxic effects of ZOL in osteosarcoma cells were mediated by the mevalonate pathway. While treatment of osteosarcoma cells with the chemotherapeutic agents doxorubicin or etoposide decreased cell viability, combination of these agents with ZOL did not significantly augment apoptosis in any of the cell lines tested. These observations suggest that ZOL has direct effects on the proliferation and survival of osteosarcoma cells in vitro, which has implications for future therapy of osteosarcoma.

The recent detection of perfluorinated compounds (PFCs) in wildlife from even remote locations has spurred interest in the environmental occurrence and effects of these chemicals. While the global distribution of PFCs is increasingly understood, there is still little information available on their effects on wildlife. The amphiphillic nature of PFCs suggests that their effects could be primarily on cell membranes. In this study we measured the effects of PFCs on membrane fluidity and mitochondrial membrane potential using flow cytometry and effects on membrane permeability using cell bioassay procedures (H4IIE, MCF-7, PLHC-1). Of the PFCs tested, only perfluorooctane sulfonic acid (PFOS) increased the permeability of cell membranes to the hydrophobic ligands used. Three PFCs were tested in the membrane fluidity assay: PFOS, perfluorohexane sulfonic acid (PFHS), and perfluorobutane sulfonic acid (PFBS). PFOS increased membrane fluidity in fish leukocytes in a dose-dependent fashion, while PFHS and PFBS had no effect in the concentration range tested. The lowest effective concentrations for the membrane fluidity effects of PFOS were 5-15 mg/l. Effects on mitochondrial membrane potential occurred in the same concentration range as effects on membrane fluidity. This suggests that PFOS effects membrane properties at concentrations below those associated with other adverse effects.