Expression of β-nerve growth factor mRNA in rat glioma cells and astrocytes from rat brain

AbstractA 50-base synthetic oligodeoxynucleotide complementary to a portion of mouse nerve growth factor (NGF) mRNA was used as a probe for analysis of the expression of NGF gene. Northern blot analysis showed the presence of a major 1.3 kb transcript, which was identical in size to mouse NGF mRNA, in both C6Bu1 cells and rat astrocytes cultured from newborn rat brain. Further, the rearrangement of DNA sequence in and around the NGF gene locus of C6Bu1 cells was not detected by Southern blot analysis. These results indicate the expression of NGF mRNA in both C6Bu1 cells and astrocytes from rat brain, suggesting that astrocytes may produce NGF protein in the rat brain, especially in developing rat brain

Role of the Aromatic Ring of Tyr43 in Tetraheme Cytochrome c(3) from Desulfovibrio vulgaris Miyazaki F

Tyrosine 43 is positioned parallel to the fifth heme axial ligand, His34, of heme 1 in the tetraheme cytochrome c(3). The replacement of tyrosine with leucine increased the redox potential of heme 1 by 44 and 35 mV at the first and last reduction steps, respectively; its effects on the other hemes are small. In contrast, the Y43F mutation hardly changed the potentials. It shows that the aromatic ring at this position contributes to lowering the redox potential of heme 1 locally, although this cannot be the major contribution to the extremely low redox potentials of cytochrome c(3). Furthermore, temperature-dependent line-width broadening in partially reduced samples established that the aromatic ring at position 43 participates in the control of the kinetics of intramolecular electron transfer. The rate of reduction of Y43L cytochrome c(3) by 5-deazariboflavin semiquinone under partially reduced conditions was significantly different from that of the wild type in the last stage of the reduction, supporting the involvement of Tyr43 in regulation of reduction kinetics. The mutation of Y43L, however, did not induce a significant change in the crystal structure

疾患活動性の高い重症筋無力症患者の胸腺ではプラズマブラストが増加している

Background and Objectives To investigate intrathymic B lymphopoiesis in patients with myasthenia gravis (MG) and explore thymus pathology associated with clinical impact. Methods Thymic lymphocytes from 15 young patients without MG, 22 adult patients without MG, 14 patients with MG without thymoma, and 11 patients with MG with thymoma were subjected to flow cytometry analysis of T follicular helper (Tfh), naive B, memory B, plasmablasts, CD19+B220high thymic B cells, B-cell activating factor receptor, and C-X-C chemokine receptor 5 (CXCR5). Peripheral blood mononuclear cells of 16 healthy subjects and 21 untreated patients with MG were also analyzed. Immunologic values were compared, and correlations between relevant values and clinical parameters were evaluated. Results The frequencies of circulating and intrathymic plasmablasts were significantly higher in patients with MG than controls. On the other hand, the frequency of CD19+B220high thymic B cells was not increased in MG thymus. We observed a significant increase in CXCR5 expression on plasmablasts in MG thymus and an increased frequency of intrathymic plasmablasts that was correlated with preoperative disease activity. The frequency of intrathymic Tfh cells was significantly lower in patients who received immunosuppressive (IS) therapy than those without IS therapy. However, there was no significant difference in the frequency of intrathymic plasmablasts irrespective of IS therapy. Discussion Our findings confirmed a correlation between increased frequency of intrathymic plasmablasts and disease activity before thymectomy. We postulate that activated intrathymic plasmablasts endow pathogenic capacity in MG

Spine Formation Pattern of Adult-Born Neurons Is Differentially Modulated by the Induction Timing and Location of Hippocampal Plasticity

<div><p>In the adult hippocampus dentate gyrus (DG), newly born neurons are functionally integrated into existing circuits and play important roles in hippocampus-dependent memory. However, it remains unclear how neural plasticity regulates the integration pattern of new neurons into preexisting circuits. Because dendritic spines are major postsynaptic sites for excitatory inputs, spines of new neurons were visualized by retrovirus-mediated labeling to evaluate integration. Long-term potentiation (LTP) was induced at 12, 16, or 21 days postinfection (dpi), at which time new neurons have no, few, or many spines, respectively. The spine expression patterns were investigated at one or two weeks after LTP induction. Induction at 12 dpi increased later spinogenesis, although the new neurons at 12 dpi didn’t respond to the stimulus for LTP induction. Induction at 21 dpi transiently mediated spine enlargement. Surprisingly, LTP induction at 16 dpi reduced the spine density of new neurons. All LTP-mediated changes specifically appeared within the LTP–induced layer. Therefore, neural plasticity differentially regulates the integration of new neurons into the activated circuit, dependent on their developmental stage. Consequently, new neurons at different developmental stages may play distinct roles in processing the acquired information by modulating the connectivity of activated circuits via their integration.</p> </div

NMDAR activity during HFS is required for LTP-mediated changes in the spinogenesis of new neurons.

<p>(<b>A</b>) Experimental schedule for CPP pretreatment. Pretreatment with CPP i.p. blocks LTP of PS amplitude in the DG, as previously described (Kitamura <i>et al</i>. 2009). Insets are samples of evoked field potential traces recorded at −1, 1, and 7 days post-HFS. (<b>B</b>) PS amplitude of rats used in this study at 1 day before, 1 day after, and 7 days after the HFS(500) delivery (−1 d, 1 d, and 7 d, respectively). CPP (10 mg/kg) was injected i.p. 2 h before the initiation of HFS delivery. (<b>C</b>) At 12 dpi, CPP was injected i.p. 2 h before HFS(500). Representative z-stack images of dendritic segments of new neurons at 28 dpi in control (<b>C1, C3</b>) and HFS-treated hemispheres (<b>C2, C4</b>) with CPP i.p. administration. (<b>C1, 2</b>) and (<b>C3, 4</b>) represent micrographs of OML and MML, respectively. Scale bars for (<b>C</b>), (<b>F</b>), and (<b>I</b>), 2 µm. (<b>F</b>) At 16 dpi, CPP was injected i.p. 2 h before HFS(500). Representative z-stack images of dendritic segments of new neurons at 28 dpi in control (<b>F1, F3</b>) and of HFS-treated hemispheres (<b>F2, F4</b>) with CPP i.p. administration. (<b>F1, 2</b>) and (<b>F3, 4</b>) represent micrographs of OML and MML, respectively. (<b>I</b>) At 21 dpi, CPP was injected i.p. 2 h before HFS(500). Representative z-stack images of dendritic segments of new neurons at 28 dpi observed in control (<b>I1, 3</b>) and HFS-treated hemispheres (<b>I2, 4</b>) with CPP i.p. administration. (<b>I1, 2</b>) and (<b>I3, 4</b>) represent micrographs of OML and MML, respectively. Dendritic segments within the HFS-delivered layer are indicated by blue characters (<b>C4, F4, and I4</b>). (<b>D</b>), (<b>G</b>), (<b>J</b>) Spine number per 10-µm dendritic fragment in each layer is graphed. (<b>E</b>), (<b>H</b>), (<b>K</b>) Averages cross-sectional area of spines is indicated. (<b>D, E</b><i>)</i> Data from new neurons treated with CPP and HFS delivery at 12 dpi. Dendritic fragments for spine analyses: control hemisphere, n = 19, HFS hemisphere, n = 19 from 3 animals. (<b>G, H</b>) Data from new neurons treated with CPP and HFS delivery at 16 dpi. Dendritic fragments for spine analyses: control hemisphere, n = 14, HFS hemisphere, n = 17 from 3 animals. (<b>J, K</b>) Data from new neurons treated with CPP and HFS delivery at 21 dpi. Dendritic fragments for spine analyses: control hemisphere, n = 19, HFS hemisphere, n = 19 from 3 animals. Data from the HFS-delivered layer are indicated by blue color in each graph. n.s. indicates no significant variance.</p

MML-LTP induction at 12 dpi enhances the later spinogenesis of new neurons specifically in MML.

<p>(<b>A</b>) Experimental schedules for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045270#pone-0045270-g002" target="_blank">Figure 2</a>. Insets are samples of evoked field potential traces that are recorded at pre-HFS, 1 day, and 16 days post-HFS. (<b>B</b>) PS amplitudes of the DG obtained from rats used for experiments in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045270#pone-0045270-g002" target="_blank">Figure 2</a>. Pre- and post-HFS delivery are indicated by “pre” and “post”, respectively. (<b>C</b>) LTP induction changes F-actin content in the DG ML. Unilateral HFS(500) was delivered to the MPP at 12 dpi, and brains were dissected at 28 dpi. DG of control hemisphere (<b>C1</b>) and LTP hemisphere (<b>C2</b>). F-actin signal (red) was visualized by phalloidin-tetramethyl rhodamine iso-thiocyanate (TRITC) staining. DG subregions are indicated in (<b>C2</b>). IML, inner ML. (<b>D</b>) Presynaptic content identified by synaptophysin signal are unchanged by MML LTP induction. Fluorescence micrographs with synaptophysin (red) in control (<b>D1</b>) and LTP (<b>D2</b>) DG. Nuclear signal is shown in blue (DRAQ5). Scale bars for (<b>C</b>) and (<b>D</b>), 50 µm. (<b>E, F</b><i>)</i> Representative z-stack images of dendritic segments of new neurons at 28 dpi in control (<b>E1, F1</b>) and LTP hemispheres (<b>E2, F2</b>). New neurons were visualized with GFP-actin. (<b>E1, 2</b>) and (<b>F1, 2</b>) represent micrographs of OML and MML, respectively. Therefore, a dendritic segment within the LTP-induced layer is depicted in (<b>F2</b>) only, indicated by red characters. Scale bar, 2 µm. (<b>G</b>) F-actin content significantly increases in MML compared with ipsilateral IML and OML and contralateral MML. Graphs show average intensity of F-actin in each DG layer in arbitrary units (AU). *, <i>P</i><0.05 from Student’s t-test. (<b>H</b>) Synaptophysin expression is unchanged by MML LTP. Graph shows MML-to-OML ratio (MML/OML) of average synaptophysin intensity in control and LTP hemispheres. n.s. indicates no significant difference. (<b>I</b>) Spine density within the LTP-induced layer significantly increases compared with other layers. Spine number per 10-µm dendritic fragment is shown in the graph. (<b>J</b>) The graph shows MML-to-OML ratio (MML/OML) of spine density in control and LTP hemispheres, with <i>P</i> values from Student’s t-test. (<b>K</b>) MML LTP induction enlarges spines in the MML. Average cross-sectional area of spines, an indicator of spine size, is graphed. (<b>G–K</b>) Dendritic fragments for spine analyses: control hemisphere, n = 22, LTP hemisphere, n = 22 from 3 animals. Data from the LTP-induced layer are indicated by red color in each graph. <i>P</i> values from post-hoc Fisher’s and Scheffe’s test are shown in (<b>I</b>), (<b>K</b>) and (<b>G</b>), respectively.</p

Spinogenesis of new neurons during the first 4 weeks after birth in adult DG.

<p>(<b>A</b>) Anatomical organization of the entorhinal-hippocampal DG pathway. Abbreviations: Rec, recording electrode; Stim, stimulating electrode; MPP and LPP, medial and lateral perforant pathway, respectively; MML and OML, middle and outer molecular layer, respectively; GCL, granule cell layer. (<b>B</b>) Side views photos showing needle tip, shaped by electrical grinder, of Hamilton syringes used for RV injection, at 90 degree orientations with respect to each other. (<b>C</b>) Representative z-stack images of morphologies and dendritic segments from newly born neurons at 12, 16, 18, 21, and 28 dpi. New neurons were identified by RV-mediated labeling with GFP-actin (green). Blue indicates nuclear distribution of individual cells (DRAQ5 staining). ML, molecular layer; GCL, granule cell layer. Selected regions in low magnification images (within squares) at 12, 16, and 18 dpi are shown below in high magnification. Scale bars: <b>a-c</b>, 50 µm; <b>d</b>, 5 µm; <b>e-j</b>, 2 µm. (<b>D</b>) Spines of new neurons are contacted by presynaptic terminals. Z-stack images are observations of dendritic fragments from discrete cells at 21 dpi. GFP-actin signal allows visualization of spines (green). The presynaptic marker synaptophysin is shown in red. Nearly all spines labeled with GFP-actin contact presynaptic terminals. Scale bar, 2 µm. (<b>E</b>) Immunoelectron microscopy showed synapse formation of GFP-actin-positive spines on 28 dpi neurons as defined by containing postsynaptic density and contacting with synaptic vesicles containing structure. <b>a</b> and <b>b</b>, typical images of dendritic spines of new neurons. <b>c</b>, Images of a filopodial protrusion were taken by tilting function (left, middle, and right photo: −50°, 0°, and +40°, respectively). Scale bar, 0.5 µm. (<b>F</b>) Density of dendritic spines on new neurons labeled with GFP-actin-RV. Line graph shows the density of protrusions on dendritic fragments of new neurons at 12, 16, 18, and 28 dpi. The protrusions density is expressed as number of protrusions per 10-µm dendritic length. (<b>G-J</b>) HFS(500)-mediated Zif268 expression in new neurons at 12 and 28 dpi. (<b>G</b>) Experimental schedule. HFS(500) was unilaterally delivered to MPP, and brains were dissected at 1 h after the initiation of HFS. (<b>H</b>) Immunohistochemistry with anti-Zif268 antibody. Left, HP of control hemisphere; right, ipsilateral hemisphere treated with HFS. Scale bar, 0.5 mm. (<b>I</b>) Triple staining for Zif268 (red), GFP (green), and DAPI (nucleus, blue). Arrowheads in GFP/Zif268 and Nucleus/Zif268 photos in each panel indicate the soma of the same GFP-labeled new neurons. Scale bar, 10 µm. (<b>J</b>) Percentage of Zif268-positive new neurons in control and HFS-delivered DG. At 12 dpi, Zif268⁺ and GFP⁺ double-positive cells were not detected (n.d.). At 12 dpi, n = 3 animals; control hemisphere, n = 15, 12, and 17 GFP+ cells in each animal; HFS-delivered hemisphere, n = 13, 12, and 7 GFP+ cells in each animal. At 28 dpi, n = 3 animals; control hemisphere, n = 19, 4, and 18 GFP+ cells in each animal; HFS-delivered hemisphere, n = 22, 8, and 15 GFP+ cells in each animal. <i>P</i> values from Student’s <i>t</i>-test are shown in the graph.</p