Mechanistic studies on the intramolecular one-electron transfer between the two flavins in the human endothelial NOS reductase domain

The object of this study was to clarify the mechanism of electron transfer in the human endothelial nitric oxide synthase (eNOS) reductase domain using recombinant eNOS reductase domains; the FAD/NADPH domain containing FAD- and NADPH-binding sites and the FAD/FMN domain containing FAD/NADPH-, FMN-, and a calmodulin-binding sites. In the presence of molecular oxygen or menadione, the reduced FAD/NADPH domain is oxidized via the neutral (blue) semiquinone (FADH(*)), which has a characteristic absorption peak at 520 nm. The FAD/NADPH and FAD/FMN domains have high activity for ferricyanide, but the FAD/FMN domain has low activity for cytochrome c. In the presence or absence of calcium/calmodulin (Ca(2+)/CaM), reduction of the oxidized flavins (FAD-FMN) and air-stable semiquinone (FAD-FMNH(*)) with NADPH occurred in at least two phases in the absorbance change at 457nm. In the presence of Ca(2+)/CaM, the reduction rate of both phases was significantly increased. In contrast, an absorbance change at 596nm gradually increased in two phases, but the rate of the fast phase was decreased by approximately 50% of that in the presence of Ca(2+)/CaM. The air-stable semiquinone form was rapidly reduced by NADPH, but a significant absorbance change at 520 nm was not observed. These findings indicate that the conversion of FADH(2)-FMNH(*) to FADH(*)-FMNH(2) is unfavorable. Reduction of the FAD moiety is activated by CaM, but the formation rate of the active intermediate, FADH(*)-FMNH(2) is extremely low. These events could cause a lowering of enzyme activity in the catalytic cycle.     

Design of a New Energy‐Harvesting Electrochromic Window Based on an Organic Polymeric Dye,a Cobalt Couple,and PProDOT‐Me2

A new design for an energy‐harvesting electrochromic window (EH‐ECW) based on the fusion of two technologies, organic electrochromic windows and dye‐sensitized solar cells (DSSCs), is presented. Unlike other power‐generating smart windows, such as photoelectrochromic devices that are passive and only contain two states (i.e., a closed‐circuit colored state and an open‐circuit bleaching state), EH‐ECW allows active tuning of the transmittance by varying the applied potential and it functions as a photovoltaic cell based on a DSSC. The resulting device demonstrates a fast switching rate of 1 s in both the bleaching and coloring processes through the use of an electrochromic polymer as a counter electrode layer. To increase the transmittance of the device, a cobalt redox couple and a light‐colored, yet efficient, organic dye are used. The organic dye contains a polymeric structure that contributes to the high cyclic stability. The device exhibits a power conversion efficiency (PCE) of 4.5% (100 mW cm^‐2) under AM 1.5 irradiation, a change in transmittance of 34% upon applied potential, and shows only 3% degradation in the PCE after 5000 cycles.     

Inference of S-system models of genetic networks using a cooperative coevolutionary algorithm

MOTIVATION: To resolve the high-dimensionality of the genetic network inference problem in the S-system model, a problem decomposition strategy has been proposed. While this strategy certainly shows promise, it cannot provide a model readily applicable to the computational simulation of the genetic network when the given time-series data contain measurement noise. This is a significant limitation of the problem decomposition, given that our analysis and understanding of the genetic network depend on the computational simulation. RESULTS: We propose a new method for inferring S-system models of large-scale genetic networks. The proposed method is based on the problem decomposition strategy and a cooperative coevolutionary algorithm. As the subproblems divided by the problem decomposition strategy are solved simultaneously using the cooperative coevolutionary algorithm, the proposed method can be used to infer any S-system model ready for computational simulation. To verify the effectiveness of the proposed method, we apply it to two artificial genetic network inference problems. Finally, the proposed method is used to analyze the actual DNA microarray data.     

Nicotine affects mineralized nodule formation by the human osteosarcoma cell line Saos-2

Several in vitro and in vivo studies have indicated that tobacco smoking may be an important risk factor for the development and severity of inflammatory periodontal disease. In the present study, we examined the effect of nicotine on cell proliferation, alkaline phosphatase (ALPase) activity, mineralized nodule formation, and the expression of extracellular matrix proteins in the human osteosarcoma cell line Saos-2. The cells were cultured with Dulbecco's modified Eagle medium containing 10% fetal bovine serum with 0, 10(-4) M, and 10(-3) M nicotine for up to 14 days. Mineralized nodule formation was examined by alizarin red staining, and the calcium content in mineralized nodules was determined using a calcium E-test kit. The expression of extracellular matrix proteins was estimated by determining the levels of their mRNAs using the real-time polymerase chain reaction. Mineralized nodule formation and calcium content in mineralized nodules were remarkably suppressed by nicotine on days 10 and 14 of culture, respectively. ALPase activity as well as type I collagen and osteopontin expression also decreased in the presence of nicotine after 5, 10, and 14 days of culture, respectively. By contrast, the amount of bone sialoprotein increased during 14 days of culture with nicotine. These results suggest that nicotine suppresses osteogenesis through a decrease in ALPase and type I collagen production by osteoblasts.     

Flotillin-1 regulates IgE receptor-mediated signaling in rat basophilic leukemia (RBL-2H3) cells

Cross-linking of high-affinity IgE receptors by multivalent Ag on mast cells (rat basophilic leukemia (RBL)-2H3) induces the phosphorylation of ITAM motifs of an IgE receptor by Src family tyrosine kinase, Lyn. The phosphorylation of IgE receptors is followed by a series of intracellular signals, such as Ca(2+) mobilization, MAPK activation, and degranulation. Therefore, Lyn is a key molecule in the activation of mast cells, but the molecular mechanisms for the activation of Lyn are still unclear. Recently, it is suggested that the localization of Lyn in lipid rafts is critical for its activation in several cell lines, although the precise mechanism is still unknown. In this study, we found that flotillin-1, which is localized in lipid rafts, is involved in the process of Lyn activation. We obtained flotillin-1 knockdown (KD)(2) rat basophilic leukemia (RBL)-2H3 cells, which express a low level of flotillin-1. In the flotillin-1 KD cells, we observed a significant decrease in Ca(2+) mobilization, the phosphorylation of ERKs, tyrosine phosphorylation of the gamma-subunit of IgE receptor, and IgE receptor-mediated degranulation. We also found that flotillin-1 is constitutively associated with Lyn in lipid rafts in RBL-2H3 cells, and Ag stimulation induced the augmentation of flotillin-1 binding to Lyn, resulting in enhancement of kinase activity of Lyn. These results suggest that flotillin-1 is an essential molecule in IgE receptor-mediated mast cell activation, and regulates the kinase activity of Lyn in lipid rafts.     

Comparison of cell lines for stable production of fucose-negative antibodies with enhanced ADCC

Several methods have been described to enhance antibody-dependent cellular cytotoxicity (ADCC) using different host cells that produce antibody with reduced levels of fucose on their carbohydrates. We compared the suitability of these methods for the serum-free fed-batch production of antibody for clinical trials and commercial uses. Recombinant anti-human CD20 chimeric IgG1-producing clones were established from host-cells that have been shown to produce more than 90% fucose-negative antibody. The cell lines were a FUT8 (alpha-1,6-fucosyltransferase) knockout Chinese hamster ovary (CHO) cell line, Ms704, and two Lens culinaris agglutinin (LCA)-resistant cell lines, one derived from a variant CHO line, Lec13 and the other from a rat hybridoma cell line, YB2/0. The amount of fucose-negative antibody produced by Lec13 and YB2/0 significantly decreased with the culture. The increase in fucosylation was due to remaining synthesis of GDP-fucose via de novo pathway for the CHO line and the elevation of FUT8 expression by the YB2/0 cells. In contrast, Ms704 cells stably produced fucose-negative antibody with a consistent carbohydrate structure until the end of the culture. The productivity of the Ms704 cells reached 1.76 g/L with a specific production rate (SPR) of 29 pg/cell/day for 17 days in serum-free fed-batch culture using a 1 L spinner bioreactor. Our results demonstrate that FUT8 knockout has the essential characteristics of host cells for robust manufacture of fucose-negative therapeutic antibodies with enhanced ADCC.     

Dietary troglitazone decreases oxidative stress in early stage type II diabetic rats

Oxidative stress is involved in the initiation and development of atherosclerosis in diabetes. We tested the hypothesis that oxidative stress is already increased in early stage type II diabetes, and that troglitazone may prevent the increase. Three groups of 20 week old rats were studied: untreated Otsuka Long-Evans Tokushima Fatty (OLETF) rats, as an animal model of type II diabetes, OLETF rats treated with troglitazone, and control Long-Evans Tokushima Otsuka (LETO) rats. Plasma lipid hydroperoxides (LOOH) concentration, as an indication of lipid peroxidation, and superoxide dismutase (SOD) activity in the thoracic aorta were measured. Plasma LOOH concentration was significantly higher in non-treated OLETF rats compared to LETO rats and treatment with troglitazone completely prevented this increase. SOD activity was significantly decreased in non-treated OLETF rats compared to LETO rats and troglitazone attenuated the diminution of it. These observations demonstrate oxidative stress is already increased in the early stage of type II diabetes and we confirmed troglitazone has the effect of an antioxidant in vivo.     

Comparison of urine cytology between the ileal conduit and Indiana pouch

OBJECTIVE: To compare the cytomorphologic features of urine obtained from two different kinds of urinary diversions constructed after total bladder resection. STUDY DESIGN: The smears of urine from 11 ileal conduits and 6 Indiana pouches were evaluated. All patients underwent total bladder resection due to transitional cell carcinoma (TCC) or other kinds of cancer before urine diversion. RESULTS: The cytologic features of Indiana pouch urine include degenerated, small, round cells without columnar cells derived from intestinal epithelium. In ileal conduit urine, well-preserved columnar cells and degenerated, small, round cells were frequently observed. The columnar cells in ileal conduit urine exhibited cytologic features that should be distinguished from TCC cells. CONCLUSION: The method of reconstructing the urinary tract is important in urine cytology from urine diversions because the cytomorphologic features of urine are different between the two kinds of urinary diversions. Since columnar cells in ileal conduit urine might lead to misdiagnosis as TCC, special consideration is required to examine ileal conduit urine.     

Unique Induction of CA1 LTP Components After Intake of Theanine, an Amino Acid in Tea Leaves and its Effect on Stress Response

Theanine, γ-glutamylethylamide, is one of the major amino acid components in green tea. This study was undertaken to evaluate the effect of theanine intake on long-term potentiation (LTP) induction at hippocampal CA1 synapses and exposure to acute stress. Young rats were fed water containing 0.3% theanine after birth. Key findings: Serum corticosterone level was markedly decreased by theanine intake. Because this decrease can modify synaptic plasticity, the effect of theanine intake was examined focused on CA1 LTP induction. CA1 LTP induced by a 100-Hz tetanus for 1 s was almost the same extent in hippocampal slices from theanine-administered rats, whereas that induced by a 200-Hz tetanus for 1 s was significantly attenuated. 2-Amino-5-phosphonovalerate (APV), an N-methyl-d-aspartate (NMDA) receptor antagonist, significantly attenuated CA1 LTP induced by a 200-Hz tetanus in the control rats, but not in theanine-administered rats. Interestingly, APV completely blocked CA1 LTP induced by a 100-Hz tetanus in the control rats, while scarcely blocking it in theanine-administered rats. These results indicate that theanine intake reduces NMDA receptor-dependent CA1 LTP, while increasing NMDA receptor-independent CA1 LTP. Furthermore, neither 100-Hz tetanus-induced LTP nor 200-Hz tetanus-induced LTP was attenuated in theanine-administered rats after exposure to tail suspension stress, suggesting that the lack of NMDA receptor-dependent CA1 LTP by theanine intake is involved in ameliorating the attenuation of CA1 LTP after tail suspension. This study is the first to indicate that theanine intake modifies the mechanism of CA1 LTP induction.     

Involvement of T6 pili in biofilm formation by serotype M6 Streptococcus pyogenes

The group A streptococcus (GAS) Streptococcus pyogenes is known to cause self-limiting purulent infections in humans. The role of GAS pili in host cell adhesion and biofilm formation is likely fundamental in early colonization. Pilus genes are found in the FCT (fibronectin-binding protein, collagen-binding protein, and trypsin-resistant antigen) genomic region, which has been classified into nine subtypes based on the diversity of gene content and nucleotide sequence. Several epidemiological studies have indicated that FCT type 1 strains, including serotype M6, produce large amounts of monospecies biofilm in vitro. We examined the direct involvement of pili in biofilm formation by serotype M6 clinical isolates. In the majority of tested strains, deletion of the tee6 gene encoding pilus shaft protein T6 compromised the ability to form biofilm on an abiotic surface. Deletion of the fctX and srtB genes, which encode pilus ancillary protein and class C pilus-associated sortase, respectively, also decreased biofilm formation by a representative strain. Unexpectedly, these mutant strains showed increased bacterial aggregation compared with that of the wild-type strain. When the entire FCT type 1 pilus region was ectopically expressed in serotype M1 strain SF370, biofilm formation was promoted and autoaggregation was inhibited. These findings indicate that assembled FCT type 1 pili contribute to biofilm formation and also function as attenuators of bacterial aggregation. Taken together, our results show the potential role of FCT type 1 pili in the pathogenesis of GAS infections.