MKK6/3 and p38 MAPK pathway activation is not necessary for insulin-induced glucose uptake but regulates glucose transporter expression

p38 mitogen-activated protein kinase (MAPK), which is situated downstream of MAPK kinase (MKK) 6 and MKK3, is activated by mitogenic or stress-inducing stimuli, as well as by insulin. To clarify the role of the MKK6/3-p38 MAPK pathway in the regulation of glucose transport, dominant negative p38 MAPK and MKK6 mutants and constitutively active MKK6 and MKK3 mutants were overexpressed in 3T3-L1 adipocytes and L6 myotubes using an adenovirus-mediated transfection procedure. Constitutively active MKK6/3 mutants up-regulated GLUT1 expression and down-regulated GLUT4 expression, thereby significantly increasing basal glucose transport but diminishing transport induced by insulin. Similar effects were elicited by chronic (24 h) exposure to tumor necrosis factor alpha, interleukin-1beta, or 200 mm sorbitol, all activate the MKK6/3-p38 MAPK pathway. SB203580, a specific p38 MAPK inhibitor, attenuated these effects, further confirming that both MMK6 and MMK3 act via p38 MAPK, whereas they had no effect on the increase in glucose transport induced by a constitutively active MAPK kinase 1 (MEK1) mutant or by myristoylated Akt. In addition, suppression of p38 MAPK activation by overexpression of a dominant negative p38 MAPK or MKK6 mutant did not diminish insulin-induced glucose uptake by 3T3-L1 adipocytes. It is thus apparent that activation of p38 MAPK is not essential for insulin-induced increases in glucose uptake. Rather, p38 MAPK activation leads to a marked down-regulation of insulin-induced glucose uptake via GLUT4, which may underlie cellular stress-induced insulin resistance caused by tumor necrosis factor alpha and other factors.     

Pigment exchange of Photosystem II reaction center by chlorophyll d

Pigment exchanges among photosystem reaction centers (RCs) are useful for the identification and functional analysis of chromophores in photosynthetic organisms. Pigment replacement within the spinach Photosystem II RC was performed with Chl d derived from the oxygenic alga Acaryochloris marina, using a protocol similar to that reported previously [Gall et al. (1998) FEBS Lett 434: 88–92] based on the incubation of reaction centers with an excess of other pigments. In this study, we analyzed Chl d-modified monomeric RC which was separated from Chl d-modified dimeric RC by size-exclusion chromatography. Based on the assumption of a constant ratio of two Pheo a molecules per RC, the number of Chl a molecules in Chl d-modified monomeric RCs was found to decrease from six to four. The absorption spectrum of the Chl d-modified monomeric RC at room temperature showed a large peak at 699.5 nm originating from Chl d and a small peak at 672.5 nm orignating from Chl a. Photoaccumulation of the Pheo a⁻ in Chl d-modified monomeric RC, in the presence of sodium dithionate and methyl viologen, did not differ significantly from that in control RC, showing that the Chl d-modified monomeric RC retains its charge separation activity and photochemically active Pheo a.     

Dynamic reorganization of microfilaments and microtubules is necessary for the expression of non-host resistance in barley coleoptile cells

To show the involvement of microfilaments and microtubules in non-host resistance of barley, partially dissected coleoptiles which had been inoculated with a non-pathogen, Erysiphe pisi, were treated with several actin and tubulin inhibitors. If the coleoptiles were not treated with any of the inhibitors, the non-pathogen always failed to penetrate the coleoptile cells. However, when coleoptiles were treated with actin or tubulin polymerization or depolymerization inhibitors, the non-pathogen was able to penetrate successfully and to form haustoria in coleoptile cells of a non-host plant, barley. Actin polymerization inhibitors, cytochalasins, were more effective in causing an increase in penetration efficiency of E. pisi than tubulin inhibitors. The effects of cytochalasins depended on the kind of cytochalasin; the strength of the actin depolymerizing activity correlated significantly with the efficiency of increasing the penetration of the non-pathogen. When both actin and tubulin inhibitors were added simultaneously, the polarization of defense-related responses, such as massive cytoplasmic aggregation, deposition of papillae and accumulation of autofluorescent compounds, at fungal penetration sites was suppressed. Actin inhibitors did not affect arrangement and stability of microtubules and vice versa, and a double treatment of coleoptile cells with both microfilament and microtubule inhibitors showed an additive effect in increasing the penetration efficiency of E. pisi. Furthermore, cytochalasin A treatment allowed other non-pathogens, Colletotrichum lagenarium and Alternaria alternata, to penetrate successfully into the non-host barley cells. These results strongly suggest that microfilaments and microtubules might play important roles in the expression of non-host resistance of barley.     

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.     

Location of nucleolar organizers in animal and plant chromosomes by means of an improved N-banding technique

With an improved N-banding technique, the location of nucleolar organizing region was determined in 27 kinds of material including mammals, a marsupial, birds, amphibians, fishes, an insect and plants. In most cases the N-bands were clearly located on certain specific regions of chromosomes, such as the secondary constriction, satellite, centromere, telomere and heterochromatic segment, while in some species they were detected as very minute bodies distributed over many chromosomes. From the available cytological and biochemical data it was suggested that the N-bands represent certain structural non-histone proteins specifically linked to nucleolar organizers in various eukaryotic chromosomes.     

Smaller Fixation Target Size Is Associated with More Stable Fixation and Less Variance in Threshold Sensitivity

The aims of this randomized observational case control study were to quantify fixation behavior during standard automated perimetry (SAP) with different fixation targets and to evaluate the relationship between fixation behavior and threshold variability at each test point in healthy young participants experienced with perimetry. SAP was performed on the right eyes of 29 participants using the Octopus 900 perimeter, program 32, dynamic strategy. The fixation targets of Point, Cross, and Ring were used for SAP. Fixation behavior was recorded using a wearable eye-tracking glass. All participants underwent SAP twice with each fixation target in a random fashion. Fixation behavior was quantified by calculating the bivariate contour ellipse area (BCEA) and the frequency of deviation from the fixation target. The BCEAs (deg²) of Point, Cross, and Ring targets were 1.11, 1.46, and 2.02, respectively. In all cases, BCEA increased significantly with increasing fixation target size (p < 0.05). The logarithmic value of BCEA demonstrated the same tendency (p < 0.05). A positive correlation was identified between fixation behavior and threshold variability for the Point and Cross targets (ρ = 0.413–0.534, p < 0.05). Fixation behavior increased with increasing fixation target size. Moreover, a larger fixation behavior tended to be associated with a higher threshold variability. A small fixation target is recommended during the visual field test.     

Involvement of intracellular iron in the toxicity of oxidized low density lipoprotein to cultured endothelial cells

We evaluated the role of iron in the toxicity of oxidized low density lipoprotein (Ox-LDL) to cultured vascular endothelial cells. Exposure of the endothelial cells to Ox-LDL led to cell lysis as judged by the release of lactate dehydrogenase into the medium. The presence of deferoxamine, an iron chelator, in the reaction medium containing Ox-LDL prevented the lysis of cells by Ox-LDL. Pretreatment of the cells with deferoxamine also reduced their susceptibility to the cytotoxicity of Ox-LDL. The formation of thiobarbituric acid-reacting substances (TBARS) was observed in the cells exposed to Ox-LDL. Pretreatment of cells with deferoxamine reduced the formation of TBARS which was induced by Ox-LDL. These observations suggest that the toxicity of Ox-LDL to cultured endothelial cells involves the lipid peroxidation of cellular membrane catalyzed by iron derived from the target (endothelial) cells.