Particle Simulation of Oxidation Induced Band 3 Clustering in Human Erythrocytes

Oxidative stress mediated clustering of membrane protein band 3 plays an essential role in the clearance of damaged and aged red blood cells (RBCs) from the circulation. While a number of previous experimental studies have observed changes in band 3 distribution after oxidative treatment, the details of how these clusters are formed and how their properties change under different conditions have remained poorly understood. To address these issues, a framework that enables the simultaneous monitoring of the temporal and spatial changes following oxidation is needed. In this study, we established a novel simulation strategy that incorporates deterministic and stochastic reactions with particle reaction-diffusion processes, to model band 3 cluster formation at single molecule resolution. By integrating a kinetic model of RBC antioxidant metabolism with a model of band 3 diffusion, we developed a model that reproduces the time-dependent changes of glutathione and clustered band 3 levels, as well as band 3 distribution during oxidative treatment, observed in prior studies. We predicted that cluster formation is largely dependent on fast reverse reaction rates, strong affinity between clustering molecules, and irreversible hemichrome binding. We further predicted that under repeated oxidative perturbations, clusters tended to progressively grow and shift towards an irreversible state. Application of our model to simulate oxidation in RBCs with cytoskeletal deficiency also suggested that oxidation leads to more enhanced clustering compared to healthy RBCs. Taken together, our model enables the prediction of band 3 spatio-temporal profiles under various situations, thus providing valuable insights to potentially aid understanding mechanisms for removing senescent and premature RBCs.     

Chronic Powder Diet After Weaning Induces Sleep,Behavioral, Neuroanatomical,and Neurophysiological Changes in Mice

The purpose of this study is to clarify the effects of chronic powder diet feeding on sleep patterns and other physiological/anatomical changes in mice. C57BL/6 male mice were divided into two groups from weaning: a group fed with solid food (SD) and a group fed with powder food (PD), and sleep and physiological and anatomical changes were compared between the groups. PD exhibited less cranial bone structure development and a significant weight gain. Furthermore, these PD mice showed reduced number of neurogenesis in the hippocampus. Sleep analysis showed that PD induced attenuated diurnal sleep/wake rhythm, characterized by increased sleep during active period and decreased sleep during rest period. With food deprivation (FD), PD showed less enhancement of wake/locomotor activity compared to SD, indicating reduced food-seeking behavior during FD. These results suggest that powder feeding in mice results in a cluster of detrimental symptoms caused by abnormal energy metabolism and anatomical/neurological changes.     

Tetraploid Embryonic Stem Cells Maintain Pluripotency and Differentiation Potency into Three Germ Layers

Polyploid amphibians and fishes occur naturally in nature, while polyploid mammals do not. For example, tetraploid mouse embryos normally develop into blastocysts, but exhibit abnormalities and die soon after implantation. Thus, polyploidization is thought to be harmful during early mammalian development. However, the mechanisms through which polyploidization disrupts development are still poorly understood. In this study, we aimed to elucidate how genome duplication affects early mammalian development. To this end, we established tetraploid embryonic stem cells (TESCs) produced from the inner cell masses of tetraploid blastocysts using electrofusion of two-cell embryos in mice and studied the developmental potential of TESCs. We demonstrated that TESCs possessed essential pluripotency and differentiation potency to form teratomas, which differentiated into the three germ layers, including diploid embryonic stem cells. TESCs also contributed to the inner cell masses in aggregated chimeric blastocysts, despite the observation that tetraploid embryos fail in normal development soon after implantation in mice. In TESCs, stability after several passages, colony morphology, and alkaline phosphatase activity were similar to those of diploid ESCs. TESCs also exhibited sufficient expression and localization of pluripotent markers and retained the normal epigenetic status of relevant reprogramming factors. TESCs proliferated at a slower rate than ESCs, indicating that the difference in genomic dosage was responsible for the different growth rates. Thus, our findings suggested that mouse ESCs maintained intrinsic pluripotency and differentiation potential despite tetraploidization, providing insights into our understanding of developmental elimination in polyploid mammals.     

Curved EFC/F-BAR-domain dimers are joined end to end into a filament for membrane invagination in endocytosis

Pombe Cdc15 homology (PCH) proteins play an important role in a variety of actin-based processes, including clathrin-mediated endocytosis (CME). The defining feature of the PCH proteins is an evolutionarily conserved EFC/F-BAR domain for membrane association and tubulation. In the present study, we solved the crystal structures of the EFC domains of human FBP17 and CIP4. The structures revealed a gently curved helical-bundle dimer of approximately 220 A in length, which forms filaments through end-to-end interactions in the crystals. The curved EFC dimer fits a tubular membrane with an approximately 600 A diameter. We subsequently proposed a model in which the curved EFC filament drives tubulation. In fact, striation of tubular membranes was observed by phase-contrast cryo-transmission electron microscopy, and mutations that impaired filament formation also impaired membrane tubulation and cell membrane invagination. Furthermore, FBP17 is recruited to clathrin-coated pits in the late stage of CME, indicating its physiological role.     

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.     

Structural and mutational studies of anthocyanin malonyltransferases establish the features of BAHD enzyme catalysis

The BAHD family is a class of acyl-CoA-dependent acyltransferases that are involved in plant secondary metabolism and show a diverse range of specificities for acyl acceptors. Anthocyanin acyltransferases make up an important class of the BAHD family and catalyze the acylation of anthocyanins that are responsible for most of the red-to-blue colors of flowers. Here, we describe crystallographic and mutational studies of three similar anthocyanin malonyltransferases from red chrysanthemum petals: anthocyanidin 3-O-glucoside-6'-O-malonyltransferase (Dm3MaT1), anthocyanidin 3-O-glucoside-3', 6'-O-dimalonyltransferase (Dm3MaT2), and a homolog (Dm3MaT3). Mutational analyses revealed that seven amino acid residues in the N- and C-terminal regions are important for the differential acyl-acceptor specificity between Dm3MaT1 and Dm3MaT2. Crystallographic studies of Dm3MaT3 provided the first structure of a BAHD member, complexed with acyl-CoA, showing the detailed interactions between the enzyme and acyl-CoA molecules. The structure, combined with the results of mutational analyses, allowed us to identify the acyl-acceptor binding site of anthocyanin malonyltransferases, which is structurally different from the corresponding portion of vinorine synthase, another BAHD member, thus permitting the diversity of the acyl-acceptor specificity of BAHD family to be understood.     

Substrate specificities of wild and mutated farnesyl diphosphate synthases from Bacillus stearothermophilus with artificial substrates

To determine the substrate specificities of wild and mutated types of farnesyl diphosphate (FPP) synthases from Bacillus stearothermophilus, we examined the reactivities of 8-hydroxygeranyl diphosphate (HOGPP) and 8-methoxygeranyl diphosphate (CH(3)OGPP) as allylic substrate homologs. The wild-type FPP synthase reaction of HOGPP (and CH(3)OGPP) with isopentenyl diphosphate (IPP) gave hydroxyfarnesyl- (and methoxyfarnesyl-) diphosphates that stopped at the first stage of condensation. On the other hand, with mutated type FPP synthase (Y81S), the former gave hydroxygeranylgeranyl diphosphate as the main double-condensation product together with hydroxyfarnesyl diphosphate as a single-condensation product and a small amount of hydroxygeranylfarnesyl diphosphate as a triple-condensation product. Moreover, the latter gave a double-condensation product, methoxygeranylgeranyl diphosphate, as the main product and only a trace of methoxyfarnesyl diphosphate was obtained.     

Syntheses of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid analogues modified by N-sulfonylamidino groups at the C-4 position and biological evaluation as inhibitors of human parainfluenza virus type 1

Eleven novel sialidase inhibitors 9 and 10 with an N-sulfonylamidino group at the C-4 position of Neu5Ac2en 1 against human parainfluenza virus type 1 (hPIV-1) were synthesized using copper-catalyzed three-component coupling reactions, and their inhibitory activities against hPIV-1 sialidase were studied.