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.     

Regulation of myosin II dynamics by phosphorylation and dephosphorylation of its light chain in epithelial cells

Nonmuscle myosin II, an actin-based motor protein, plays an essential role in actin cytoskeleton organization and cellular motility. Although phosphorylation of its regulatory light chain (MRLC) is known to be involved in myosin II filament assembly and motor activity in vitro, it remains unclear exactly how MRLC phosphorylation regulates myosin II dynamics in vivo. We established clones of Madin Darby canine kidney II epithelial cells expressing MRLC-enhanced green fluorescent protein or its mutants. Time-lapse imaging revealed that both phosphorylation and dephosphorylation are required for proper dynamics of myosin II. Inhibitors affecting myosin phosphorylation and MRLC mutants indicated that monophosphorylation of MRLC is required and sufficient for maintenance of stress fibers. Diphosphorylated MRLC stabilized myosin II filaments and was distributed locally in regions of stress fibers where contraction occurs, suggesting that diphosphorylation is involved in the spatial regulation of myosin II assembly and contraction. We further found that myosin phosphatase or Zipper-interacting protein kinase localizes to stress fibers depending on the activity of myosin II ATPase.     

Boss/Sev signaling from germline to soma restricts germline-stem-cell-niche formation in the anterior region of Drosophila male gonads

Drosophila germline stem cells are regulated by the somatic microenvironment, or "niche," which ensures that the stem cells can both self-renew and produce functional gametes throughout adult life. However, despite its prime importance, little is known about how niche formation is regulated during gonadal development. Here, we demonstrate that a receptor tyrosine kinase, Sevenless (Sev), is required to ensure that the niche develops in the anterior region of the male embryonic gonads. Sev is expressed in somatic cells within the posterior region of the gonads. Sev is activated by a ligand, Bride of sevenless (Boss), which is expressed by the germline, to prevent ectopic niche differentiation in the posterior gonadal somatic cells. Thus, we propose that signal transduction from germline to soma restricts expansion of the germline-stem-cell niche in the gonads.     

IKKε regulates cell elongation through recycling endosome shuttling

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Highlights? Recycling endosomes shuttle along the proximal-distal axis of growing bristles ? IKK? locally antagonizes Rab11 effector Nuf at the bristle tip by phosphorylation ? Nuf-endosome pathway is independent of actin cytoskeleton or DIAP1-caspase pathway ? Mammalian IKK-related kinases antagonize Rab11-FIP3 through phosphorylation     

Evaluation ofl-lactic acid production in batch, fed-batch, and continuous cultures ofRhizopus sp. MK-96-1196 using an airlift bioreactor

Various processes which producel-lactic acid using ammonia-tolerant mutant strain,Rhizopus sp. MK-96-1196, in a 3 L airlift bioreactor were evaluated. When the fed-batch culture was carried out by keeping the glucose concentration at 30 g/l, more than 140 g/l ofl-lactic acid was produced with a product yield of 83%. In the case of the batch culture with 200 g/l of initial glucose concentration, 121 g/L ofl-lactic acid was obtained but the low product yield based on the amount of glucose consumed. In the case of a continuous culture, 1.5 g/l/h of the volumetric productivity with a product yield of 71% was achieved at dilution rate of 0.024 h⁻¹. Basis on these results three processes were evaluated by simple variable cost estimation including carbon source, steam, and waste treatment costs. The total variable costs of the fed-batch and continuous cultures were 88% and 140%, respectively, compared to that of batch culture. The fed-batch culture with highl-lactic acid concentration and high product yield decreased variable costs, and was the best-suited for the industrial production ofl-lactic acid.     

Nuclear transfer of perchloric acid-soluble protein by endoplasmic reticulum stressors

Perchloric acid-soluble protein (PSP) is highly conserved during evolution from bacteria to mammals. Although PSP has been recognized as an inhibitor of translation and proliferation in vitro, its precise biological role has not yet been elucidated. Since we previously found similar distributions for PSP and the endoplasmic reticulum (ER) and Golgi complex, the intracellular distribution of PSP was analyzed in more detail. Immunofluorescence studies indicated that PSP co-localized with the ER and Golgi complex, since the distribution pattern of PSP was well matched to both of these organelles. An immunoelectron microscopic study revealed PSP was located not only in the cytosol but also on the surface of the outer ER membrane. Since PSP was present on the ER, we speculated that it may be associated with ER function. Therefore, we analyzed whether or not the ER stress response, which is one of the ER functions, affected PSP expression. The results showed that various ER stressors (thapsigargin, A23187, tunicamycin, brefeldin A, and cisplatin) provoked a dramatic change in the localization of PSP from outside of the nucleus to inside the nucleus within 3 h. Moreover, the ER stressors induced PSP expression. These results suggest that PSP is involved in the cellular response to ER stressors, and that the change in localization of PSP from the ER to the nucleus may be associated with ER stress responses.     

Rho localization in cells and tissues

Rho family small GTPases regulate cytoskeletal organization. Although their spatiotemporal activities appear to be important for cellular morphogenesis, there has been little characterization of the localization of Rho family GTPases in cells and tissues. Here we show precise localization of Rho subfamily proteins in mammalian cultured cells and tissues through evaluation of anti-Rho antibodies and fixation protocols. Although Rho is not a structural protein but functions as a switching molecule, it often localizes at several distinct domains or structures of cells. In cultured epithelial cells, Rho was highly accumulated at lateral membranes. However, in fibroblastic cells, Rho appeared to be distributed evenly in the cytoplasm. Rho concentration at the cleavage furrow at cytokinesis was generally observed. In A431 cells, Rho translocation from the cytoplasm to elongating microvilli at the apical membrane within 30 s after EGF stimulation was clearly demonstrated. Also, Myc- or GFP-tagged RhoA did not always reflect the localization of endogenous Rho, indicating a drawback of protein-tagging methods for localization research. In mouse tissues, Rho localization differed depending on cell type, probably reflecting the functional differences of each cell type.