Effects of 10 years of fencing under a gap and closed canopy on the regeneration of tree seedlings in an old-growth Japanese fir (<i>Abies firma</i>) forest overbrowsed by sika deer

<p>We report the effects of 10 years of fencing under a gap and under a closed canopy on the regeneration of tree seedlings in an old-growth fir forest overbrowsed by sika deer in the Tanzawa Mountains. We examined tree seedling emergence and growth of 10 major species over 10 years at four plots: inside and outside a deer exclosure under a gap and under a closed canopy (the plots were named “GAPF”, “GAP”, “CCF” and “CC”, respectively). Seedlings of <i>Abies firma</i>, <i>Acer palmatum</i> and <i>Zelkova serrata</i> were noted at all four plots during the initial investigation. Seedlings of five pioneer species emerged in GAPF and GAP, but had showed almost no emergence in CC and CCF. Seedlings of 10 target species showed the greatest tendency toward survival at GAPF among the plots. The survival of six species’ seedlings was significantly higher (<i>p</i> < 0.0487) at GAPF than at CCF. The height of most seedlings was <20 cm at first measurement. After 10 years, the maximum heights of 10 species showed that all species grew taller at GAPF but scarcely grew at the other plots. The height distribution at GAPF showed that pioneer species dominated >150 cm, while <i>A. firma, A. palmatum</i> and <i>Z. serrata</i> were mainly <120 cm. These results suggest that the 10 species were able to survive and grow most successfully in the gap without sika deer and that the construction of a deer exclosure just after gap formation was effective in promoting the regeneration of the overbrowsed forest.</p

Oligo(ethylene glycol)-modified β-cyclodextrin-based polyrotaxanes for simultaneously modulating solubility and cellular internalization efficiency

<p>We developed stimuli-labile polyrotaxanes (PRXs) composed of β-cyclodextrin (β-CD), Pluronic as an axle polymer, and acid-cleavable <i>N</i>-triphenylmethyl groups as bulky stopper molecules, and found that the PRXs are potent therapeutics for Niemann-Pick type C disease, because the PRX can effectively reduce intracellular cholesterol through the intracellular release of threaded β-CDs. In general, the PRXs need to be chemically modified with hydrophilic functional groups because PRXs are not soluble in aqueous media. Herein, four series of oligo(ethylene glycol)s (OEGs) with different ethylene glycol repeating unit (2 or 3) and chemical structure of OEG terminal (hydroxy or methoxy) were modified onto the threaded β-CDs in PRX. The effects of the structure of OEG on the aqueous solubility, toxicity, and cellular internalization efficiency of OEG-modified PRXs were investigated to optimize the chemical structure of OEG. The hydroxy-terminated OEG-modified PRXs showed excellent solubility in aqueous media and no toxicity, regardless of the number of ethylene glycol repeating units. In the case of the methoxy-terminated OEG-modified PRXs, sufficient solubility in aqueous media and negligible toxicity were observed when the number of ethylene glycol repeating units was 3, while low solubility and toxicity were observed when the ethylene glycol repeating unit was 2. Additionally, cellular uptake levels of methoxy-terminated OEG-modified PRXs in RAW264.7 cells were higher than those of hydroxy-terminated OEG-modified PRXs. Consequently, the chemical structure of the OEG strongly affects the chemical and biological properties of the PRXs, and that a methoxy-terminated OEG with 3 ethylene glycol repeating units is the most preferable modification of PRXs, since the resultant PRX is sufficiently soluble in aqueous media, non-toxic, and possesses high cellular internalization efficiency.</p

Acid-Induced Intracellular Dissociation of β‑Cyclodextrin-Threaded Polyrotaxanes Directed toward Attenuating Phototoxicity of Bisretinoids through Promoting Excretion

In the retinal pigment epithelium of patients with age-related macular degeneration (AMD), excess <i>N</i>-retinylidene-<i>N</i>-retinylethanolamine (A2E), a dimer of all-<i>trans</i>-retinal, accumulats to induce inflammatory cytokine secretion and phototoxic effects. Therefore, the reduction of intracellular A2E is a promising approach for the prevention and treatment of AMD. In this study, acid-labile β-cyclodextrin (β-CD)-threaded polyrotaxanes (PRXs) were synthesized and investigated their effects on the removal of A2E accumulated in retinal pigment epithelium cells (ARPE-19) in comparison to nonlabile PRXs and 2-hydroxypropyl β-CD (HP-β-CD) were examined. GC-MS and HPLC studies strongly suggest that the acid-labile PRXs dissociated into their constituent molecules in cells by lysosomal acidification and threaded β-CDs were considered to be released from the PRXs. The released β-CDs formed an inclusion complex with A2E, which promoted the excretion of A2E. Indeed, the acid-labile PRXs effectively reduced intracellular A2E level at approximately a 10-fold lower concentration than HP-β-CD. Accompanied with A2E removal, the toxicity and phototoxicity of A2E were attenuated by treatment with acid-labile PRXs. Because the nonlabile PRX failed to reduce intracellular A2E level and attenuate phototoxicity, intracellular release of threaded β-CDs from the acid-labile PRX might contribute to reducing intracellular A2E. We conclude that acid-labile PRXs are promising candidates for the treatment of macular diseases through the removal of toxic metabolites

The effect of methacryloyl group of HEMA on the expression of CD54 and CD86.

<p>Relative viability of (A) and expression levels of CD54 (B) and CD86 (C) in THP-1 cells treated with HEMA (closed triangles) or HEIB (open triangles) at various concentrations for 24 h. The plots for HEMA were taken from Figure 2. The data are expressed as the means ± S.D. (n=3) (*<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.005, ****<i>p</i> < 0.001).</p

ROS production in THP-1 cells by the treatment with HEMA.

<p>Relative ROS levels in THP-1 cells treated with HEMA (A) or HEIB (B) at various concentrations for 24 h. The data are expressed as the means ± S.D. (n=3) (*<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.005, ****<i>p</i> < 0.001).</p

Expression of CD54 and CD86 in THP-1 cells by the treatment with HEMA, MMA and NiSO₄.

<p>Relative viability of (A) and expression levels of CD54 (B) and CD86 (C) in THP-1 cells treated with NiSO₄ (open circles), HEMA (closed triangles), or MMA (closed squares) at various concentrations for 24 h. The data are expressed as the means ± S.D. (n=3) (*<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.005, ****<i>p</i> < 0.001).</p

Expression of CD54 and CD86 in THP-1 cells by the treatment with water-soluble PHEMA oligomer.

<p>Relative viability of (A) and expression levels of CD54 (B) and CD86 (C) in THP-1 cells treated with HEMA (closed triangles), PHEMA15 (open triangles ), or PHEMA35 (closed diamonds) at various concentrations for 24 h. The plots for HEMA were taken from Figure 2. The data are expressed as the means ± S.D. (n=3) (*<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.005, ****<i>p</i> < 0.001).</p

Both Neurons and Astrocytes Exhibited Tetrodotoxin-Resistant Metabotropic Glutamate Receptor-Dependent Spontaneous Slow Ca²⁺ Oscillations in Striatum

<div><p>The striatum plays an important role in linking cortical activity to basal ganglia outputs. Group I metabotropic glutamate receptors (mGluRs) are densely expressed in the medium spiny projection neurons and may be a therapeutic target for Parkinson's disease. The group I mGluRs are known to modulate the intracellular Ca²⁺ signaling. To characterize Ca²⁺ signaling in striatal cells, spontaneous cytoplasmic Ca²⁺ transients were examined in acute slice preparations from transgenic mice expressing green fluorescent protein (GFP) in the astrocytes. In both the GFP-negative cells (putative-neurons) and astrocytes of the striatum, spontaneous slow and long-lasting intracellular Ca²⁺ transients (referred to as slow Ca²⁺ oscillations), which lasted up to approximately 200 s, were found. Neither the inhibition of action potentials nor ionotropic glutamate receptors blocked the slow Ca²⁺ oscillation. Depletion of the intracellular Ca²⁺ store and the blockade of inositol 1,4,5-trisphosphate receptors greatly reduced the transient rate of the slow Ca²⁺ oscillation, and the application of an antagonist against mGluR5 also blocked the slow Ca²⁺ oscillation in both putative-neurons and astrocytes. Thus, the mGluR5-inositol 1,4,5-trisphosphate signal cascade is the primary contributor to the slow Ca²⁺ oscillation in both putative-neurons and astrocytes. The slow Ca²⁺ oscillation features multicellular synchrony, and both putative-neurons and astrocytes participate in the synchronous activity. Therefore, the mGluR5-dependent slow Ca²⁺ oscillation may involve in the neuron-glia interaction in the striatum.</p></div

GFP was expressed only in astrocytes, and GFP-negative cells in GFAP-GFP mice were mainly neurons.

<p>A1, 2, Expression of GFP (green) and S-100 (red), a marker of astrocytes, in striatal slices of the GFAP-GFP mouse. Most S-100-positive cells expressed GFP. The proportions of cells positive for both GFP and S-100, for only S-100, and for only GFP were 62.4±1.8%, 30.3±1.9%, and 7.2±1.1%, respectively (n = 5 slices, total cell number  = 1509 cells). B1, 2, Expression of GFP (green) and NeuN (red), a marker of neurons. Few double-stained cells were observed (n = 4 slices). C1, 2, NeuN (green) and S-100 (red) expression in striatal slices of non-transgenic mice. In the striatum, fewer astrocytes were observed than neurons, and the proportion of NeuN-positive cells to S-100-positive cells was approximately 2∶1 (n = 3 slices). A2, B2, C2, The magnified images in the dashed boxes shown in A1, B1, C1, respectively. Scale bars, 50 µm.</p

Ezrin Mediates Neuritogenesis via Down-Regulation of RhoA Activity in Cultured Cortical Neurons

<div><p>Neuronal morphogenesis is implicated in neuronal function and development with rearrangement of cytoskeletal organization. Ezrin, a member of Ezrin/Radixin/Moesin (ERM) proteins links between membrane proteins and actin cytoskeleton, and contributes to maintenance of cellular function and morphology. In cultured hippocampal neurons, suppression of both radixin and moesin showed deficits in growth cone morphology and neurite extensions. Down-regulation of ezrin using siRNA caused impairment of netrin-1-induced axon outgrowth in cultured cortical neurons. However, roles of ezrin in the neuronal morphogenesis of the cultured neurons have been poorly understood. In this report, we performed detailed studies on the roles of ezrin in the cultured cortical neurons prepared from the ezrin knockdown (<i>Vil2^kd/kd</i>) mice embryo that showed a very small amount of ezrin expression compared with the wild-type (<i>Vil2^+/+</i>) neurons. Ezrin was mainly expressed in cell body in the cultured cortical neurons. We demonstrated that the cultured cortical neurons prepared from the <i>Vil2^kd/kd</i> mice embryo exhibited impairment of neuritogenesis. Moreover, we observed increased RhoA activity and phosphorylation of myosin light chain 2 (MLC2), as a downstream effector of RhoA in the <i>Vil2^kd/kd</i> neurons. In addition, inhibition of Rho kinase and myosin II rescued the impairment of neuritogenesis in the <i>Vil2^kd/kd</i> neurons. These data altogether suggest a novel role of ezrin in the neuritogenesis of the cultured cortical neurons through down-regulation of RhoA activity.</p></div