Hydroxyl Radical Formation in Skeletal Muscle of Rats with Glucocorticoid-Induced Myopathy

Steroid myopathy is a well-known adverse effect of glucocorticoids that causes muscle weakness and atrophy; however, its pathogenic mechanism is still unclear. Recently, oxidative stress was reported to contribute to steroid myopathy, but there is no report that actually attempts to measure hydroxyl radical. I developed an animal model of steroid myopathy in rat with dexamethasone (9-Fluoro−11β,17, 21-trihydroxy−16α-methylpregna−1,4-diene−3,20-dione), and measured hydroxyl radical using the salicylate trapping method. There was significant dose-dependent relation between both 2,5- and 2,3-dihydroxybenzoic acids and dexamethasone in the treated group, compared to the control group. These results suggest that hydroxyl radical plays a role in the pathogenesis of steroid myopathy.     

Blue-light-induced reorganization of the actin cytoskeleton and the avoidance response of chloroplasts in epidermal cells of<Emphasis Type="Italic"> Vallisneria gigantea</Emphasis>

In leaf epidermal cells of the aquatic angiosperm Vallisneria gigantea Graebner, high-intensity blue light induces the actin-dependent avoidance response of chloroplasts. By semi-quantitative motion analysis and phalloidin staining, time courses of the blue-light-induced changes in the mode of movement of individual chloroplasts and in the configuration of actin filaments were examined in the presence and absence of a flavoprotein inhibitor, diphenylene iodonium. In dark-adapted cells, short, thick actin bundles seemed to surround each chloroplast, which was kept motionless in the outer periclinal cytoplasm of the cells. After 10 min of irradiation with high-intensity blue light, a rapid, unidirectional movement of chloroplasts was induced, concomitant with the appearance of aggregated, straight actin bundles stretched over the outer periclinal cytoplasm. Diphenylene iodonium inhibited the avoidance response of chloroplasts, apparently by delaying a change in the mode of chloroplast movement from random sway to unidirectional migration, by suppressing the appearance of aggregated, straight actin bundles. In partially irradiated individual cells, redistribution of chloroplasts and reorganization of actin filaments occurred only in the areas exposed to blue light. From the results, we propose that the short, thick actin bundles in the vicinity of chloroplasts function to anchor the chloroplasts in dark-adapted cells, and that the aggregated, straight actin bundles organized under blue-light irradiation provide tracks for unidirectional movement of chloroplasts.Preliminary results of part of the local irradiation study have already been reported in abstract form [N. Sakurai et al. (2002) J Photosci 9:326–328].     

Victorin triggers programmed cell death and the defense response via interaction with a cell surface mediator

The host-selective toxin victorin is produced by Cochliobolus victoriae, the causal agent of victoria blight of oats. Victorin has been shown to bind to the P protein of the glycine decarboxylase complex (GDC) in mitochondria, and induce defense-related responses such as phytoalexin synthesis, extracellular alkalization and programmed cell death. However, evidence demonstrating that the GDC plays a critical role in the onset of cell death is still lacking, and the role of defense-like responses in the pathogenicity has yet to be elucidated. Here, cytofluorimetric analyses, using the fluorescein (VicFluor) or bovine serum albumin-fluorescein derivative of victorin (VicBSA), demonstrated that victorin-induced cell death occurs before these conjugates traverse the plasma membrane. As with native victorin, VicBSA clearly elicits apoptosis-like cell death, production of phytoalexin, extracellular alkalization, and generation of nitric oxide and reactive oxygen intermediates. These results suggest that the initial recognition of victorin takes place on the cell surface, not in mitochondria, and leads to the activation of a battery of victorin-induced responses. Pharmacological studies showed that extracellular alkalization is the essential regulator for both victorin- and VicBSA-induced cellular responses. We propose a model where victorin may kill the host cell by activating an HR-like response, independent of the binding to the GDC, through ion fluxes across the plasma membrane.     

Novel matrix metalloproteinase inhibitors: generation of lead compounds by the in silico fragment-based approach

Generation of structurally new matrix metalloproteinase inhibitors was successfully carried out using an in silico technique. In order to identify the small fragment interacting with residues in the S1' pocket of MMP-1 through hydrogen bonds, we performed in silico screening using the LUDI program. As a result, acetyl-L-alanyl-(N-methyl)amide (Ac-L-Ala-NHMe) was selected to link with another fragment, hydroxamic acid that interacted with catalytic zinc. By this approach, the L-glutamic acid derivative 2b was discovered to be a new type of matrix metalloproteinase inhibitor. Further transformation to reduce its peptidic nature and improve activity yielded nonpeptidic lead compounds as inhibitors of MMP-1, -2, -3, and -9.     

HMGB1 contributes to the development of acute lung injury after hemorrhage

High mobility group box 1 (HMGB1) is a novel late mediator of inflammatory responses that contributes to endotoxin-induced acute lung injury and sepsis-associated lethality. Although acute lung injury is a frequent complication of severe blood loss, the contribution of HMGB1 to organ system dysfunction in this setting has not been investigated. In this study, HMGB1 was detected in pulmonary endothelial cells and macrophages under baseline conditions. After hemorrhage, in addition to positively staining endothelial cells and macrophages, neutrophils expressing HMGB1 were present in the lungs. HMGB1 expression in the lung was found to be increased within 4 h of hemorrhage and then remained elevated for more than 72 h after blood loss. Neutrophils appeared to contribute to the increase in posthemorrhage pulmonary HMGB1 expression since no change in lung HMGB1 levels was found after hemorrhage in mice made neutropenic with cyclophosphamide. Plasma concentrations of HMGB1 also increased after hemorrhage. Blockade of HMGB1 by administration of anti-HMGB1 antibodies prevented hemorrhage-induced increases in nuclear translocation of NF-kappa B in the lungs and pulmonary levels of proinflammatory cytokines, including keratinocyte-derived chemokine, IL-6, and IL-1 beta. Similarly, both the accumulation of neutrophils in the lung as well as enhanced lung permeability were reduced when anti-HMGB1 antibodies were injected after hemorrhage. These results demonstrate that hemorrhage results in increased HMGB1 expression in the lungs, primarily through neutrophil sources, and that HMGB1 participates in hemorrhage-induced acute lung injury.     

Effect of color temperature of light sources on slow-wave sleep

In order to examine whether the spectral compositions of light source may affect sleep quality, sleep architecture under different color temperatures of light sources was evaluated. Seven healthy males were exposed to the light sources of different color temperatures (3000 K, 5000 K and 6700 K) for 6.5 h before sleep. The horizontal illuminance level was kept at 1000 lux. Subjects slept on a bed in near darkness (< 10 lux) after extinguishing the light, and polysomnograms recorded the sleep parameters. In the early phase of the sleep period, the amount of stage-4 sleep (S4-sleep) was significantly attenuated under the higher color temperature of 6700 K compared with the lower color temperature of 3000 K. Present findings suggest that light sources with higher color temperatures may affect sleep quality in a view that S4-sleep period is important for sleep quality.     

Comparison between TRF2 and TRF1 of their telomeric DNA-bound structures and DNA-binding activities

Mammalian telomeres consist of long tandem arrays of double-stranded telomeric TTAGGG repeats packaged by the telomeric DNA-binding proteins TRF1 and TRF2. Both contain a similar C-terminal Myb domain that mediates sequence-specific binding to telomeric DNA. In a DNA complex of TRF1, only the single Myb-like domain consisting of three helices can bind specifically to double-stranded telomeric DNA. TRF2 also binds to double-stranded telomeric DNA. Although the DNA binding mode of TRF2 is likely identical to that of TRF1, TRF2 plays an important role in the t-loop formation that protects the ends of telomeres. Here, to clarify the details of the double-stranded telomeric DNA-binding modes of TRF1 and TRF2, we determined the solution structure of the DNA-binding domain of human TRF2 bound to telomeric DNA; it consists of three helices, and like TRF1, the third helix recognizes TAGGG sequence in the major groove of DNA with the N-terminal arm locating in the minor groove. However, small but significant differences are observed; in contrast to the minor groove recognition of TRF1, in which an arginine residue recognizes the TT sequence, a lysine residue of TRF2 interacts with the TT part. We examined the telomeric DNA-binding activities of both DNA-binding domains of TRF1 and TRF2 and found that TRF1 binds more strongly than TRF2. Based on the structural differences of both domains, we created several mutants of the DNA-binding domain of TRF2 with stronger binding activities compared to the wild-type TRF2.     

De novo formation of left-right asymmetry by posterior tilt of nodal cilia

In the developing mouse embryo, leftward fluid flow on the ventral side of the node determines left–right (L-R) asymmetry. However, the mechanism by which the rotational movement of node cilia can generate a unidirectional flow remains hypothetical. Here we have addressed this question by motion and morphological analyses of the node cilia and by fluid dynamic model experiments. We found that the cilia stand, not perpendicular to the node surface, but tilted posteriorly. We further confirmed that such posterior tilt can produce leftward flow in model experiments. These results strongly suggest that L-R asymmetry is not the descendant of pre-existing L-R asymmetry within each cell but is generated de novo by combining three sources of spatial information: antero-posterior and dorso-ventral axes, and the chirality of ciliary movement.     

Conversion of diacetyl-C-(beta-D-glucopyranosyl)phloroglucinol to spiroketal compounds

Diacetyl-C-(beta-D-glucopyranosyl)phloroglucinol was converted by refluxing in water to spiro(benzofuran-[2H]furan) a new compound, along with spiro(benzofuran-[2H]pyran). The stereochemistry of the quaternary carbon of both spiro compounds had an S-configuration.     

Environmentally friendly C-glycosylation of phloroacetophenone with unprotected D-glucose using scandium(III) trifluoromethanesulfonate in aqueous media: key compounds for the syntheses of mono- and di-C-glucosylflavonoids

The direct C-glycosylation of phloroacetophenone with an unprotected d-glucose in aqueous media using scandium(III) trifluoromethanesulfonate (Sc(OTf)3) as the catalyst, gave mono- and bis-C-beta-glycosylic compounds in highest total yield of 81%. The second and third use of the recovered Sc(OTf)3 afforded them in total yields of 56% and 53%, respectively.