電気的形質転換法を用いた cDNA ライブラリーの作製(農芸化学部門)

電気的形質転換法を用いプラスミドDNAを導入することにより大腸菌を形質転換した。我々は高効率を得る工夫をし, この方法を用いることでコンピテントセルを用いる形質転換法(Hanahan法)では出せない高効率を得ることが可能になった。ここではこの方法で登熟期イネ胚乳及びホウレンソウ芽生えよりcDNAライブラリーの作製に成功した。To transform Escherichia coli a plasmid DNA was introduced by electroporation. We improved transformation efficiency adding several chemicals, biological materials and/or preheating of the buffer solution which was added just affer electrodischarge. The improved method gave higher transformation efficiency comparing with that of Hanahan\u27s. cDNA libraries for mRNAs appearing in the developing rice seeds and spinach seedlings were constructed using this method

Involvement of Degenerating 21.5 kDa Isoform of Myelin Basic Protein in the Pathogenesis of the Relapse in Murine Relapsing–Remitting Experimental Autoimmune Encephalomyelitis and MS Autopsied Brain

Multiple sclerosis (MS) is the chronic inflammatory demyelinating disease of the CNS. Relapsing–remitting MS (RRMS) is the most common type of MS. However, the mechanisms of relapse and remission in MS have not been fully understood. While SJL mice immunized with proteolipid protein (PLP) develop relapsing–remitting experimental autoimmune encephalomyelitis (RR-EAE), we have recently observed that some of these mice were resistant to the active induction of relapsing EAE after initial clinical and histological symptoms of EAE with a severity similar to the relapsing EAE mice. To clarify the mechanism of relapsing, we examined myelin morphology during PLP139–151-induced RR-EAE in the SJL mice. While RR-EAE mice showed an increased EAE severity (relapse) with CNS inflammation, demyelination with abnormal myelin morphology in the spinal cord, the resistant mice exhibited a milder EAE phenotype with diminished relapse. Compared with the RR-EAE mice, the resistant mice showed less CNS inflammation, demyelination, and abnormalities of the myelin structure. In addition, scanning electron microscopic (SEM) analysis with the osmium-maceration method displayed ultrastructural abnormalities of the myelin structure in the white matter of the RR-EAE spinal cord, but not in that of the resistant mice. While the intensity of myelin staining was reduced in the relapsing EAE spinal cord, immunohistochemistry and immunoblot analysis revealed that the 21.5 kDa isoform of degenerating myelin basic protein (MBP) was specifically induced in the relapsing EAE spinal cord. Taken together, the neuroinflammation-induced degenerating 21 kDa isoform of MBP sheds light on the development of abnormal myelin on the relapse of MS pathogenesis

Protein Tyrosine Phosphatase Receptor Type Z Negatively Regulates Oligodendrocyte Differentiation and Myelination

<div><h3>Background</h3><p>Fyn tyrosine kinase-mediated down-regulation of Rho activity through activation of p190RhoGAP is crucial for oligodendrocyte differentiation and myelination. Therefore, the loss of function of its counterpart protein tyrosine phosphatase (PTP) may enhance myelination during development and remyelination in demyelinating diseases. To test this hypothesis, we investigated whether Ptprz, a receptor-like PTP (RPTP) expressed abuntantly in oligodendrocyte lineage cells, is involved in this process, because we recently revealed that p190RhoGAP is a physiological substrate for Ptprz.</p> <h3>Methodology/Principal Findings</h3><p>We found an early onset of the expression of myelin basic protein (MBP), a major protein of the myelin sheath, and early initiation of myelination <em>in vivo</em> during development of the <em>Ptprz</em>-deficient mouse, as compared with the wild-type. In addition, oligodendrocytes appeared earlier in primary cultures from <em>Ptprz</em>-deficient mice than wild-type mice. Furthermore, adult <em>Ptprz</em>-deficient mice were less susceptible to experimental autoimmune encephalomyelitis (EAE) induced by active immunization with myelin/oligodendrocyte glycoprotein (MOG) peptide than were wild-type mice. After EAE was induced, the tyrosine phosphorylation of p190RhoGAP increased significantly, and the EAE-induced loss of MBP was markedly suppressed in the white matter of the spinal cord in <em>Ptprz</em>-deficient mice. Here, the number of T-cells and macrophages/microglia infiltrating into the spinal cord did not differ between the two genotypes after MOG immunization. All these findings strongly support the validity of our hypothesis.</p> <h3>Conclusions/Significance</h3><p>Ptprz plays a negative role in oligodendrocyte differentiation in early central nervous system (CNS) development and remyelination in demyelinating CNS diseases, through the dephosphorylation of substrates such as p190RhoGAP.</p> </div

Increased phosphorylation of Tyr 1105 on p190RhoGAP in the spinal cord of <i>Ptprz</i>-deficient mice after EAE induction.

<p><b><i>A</i></b>, Overall tyrosine phosphorylation patterns of total protein and expression of p190RhoGAP and Fyn in the spinal cord. The third to sixth lumbar spinal cord extracts were prepared from wild-type (+/+) and <i>Ptprz</i>-deficient mice (−/−) 35 days after MOG immunization, or non-immunized control animals, and examined by Western blotting using anti-phosphotyrosine PY20 (top), anti-p190RhoGAP (middle), and anti-Fyn (bottom) antibodies, respectively. <b><i>B</i></b>, Tyrosine phosphorylation of Tyr 1105 on p190RhoGAP. The spinal cord extracts were immunoprecipitated with anti-p190RhoGAP antibody and immunoblotted with anti-pY1105 p190RhoGAP (upper), or anti-p190RhoGAP (lower). The densitometric data for anti-pY1105 p190RhoGAP signals are presented as a percentage of the non-immunized wild-type control, and shown at the bottom. Data are the mean ± SEM (<i>n</i> = 4 pooled samples from two animals per each group). *<i>p</i><0.05 (Student's <i>t</i>-test). <b><i>C</i></b>, No siginificant differences in tyrosine phosphorylation of Fyn among the four groups. The spinal cord extracts prepared as above were immunoprecipitated with anti-Fyn antibody and immunoblotted with anti-pY420 (top), anti-pY531 (middle), or anti-Fyn (bottom). The densitometric data for anti-pY420 and anti-pY531 signals are presented as a percentage of the non-immunized wild-type control, and shown at the bottom. Data are the mean ± SEM (<i>n</i> = 4 pooled samples from two animals per group). No significant differences were detected between the two genotypes.</p

Reduced MBP loss in <i>Ptprz</i>-deficient mice after EAE induction.

<p>Anti-MBP staining of the spinal cord sections from wild-type and <i>Ptprz</i>-deficient mice 35 days after MOG immunization, or non-immunized control mice. The lower images are enlargements of the areas enclosed by squares in the upper images. Scale bars, 500 µm. The densitometric data for MBP signals are expressed as the relative change (fold-increase) compared with the non-immunized wild-type mice, and shown at the bottom. Data are the mean ± SEM (<i>n</i> = 6 for each group). **<i>p</i><0.01 (Student's <i>t</i>-test). a.u., arbitrary unit.</p

Early initiation of myelination in <i>Ptprz</i>-deficient mice.

<p><b><i>A</i></b>, <b><i>B</i></b>, Immunohistochemical analyses of MBP expression in mouse brains at postnatal day 10 (A), and 3 months old (B). Scale bars, 1 mm. The results of the densitometric analysis of MBP signals are normalized to the value for respective wild-type controls, and shown at the lower position of each panel. Data are the mean ± SEM (<i>n</i> = 3 for each group). *<i>p</i><0.05 (Student's <i>t</i>-test). a.u., arbitrary unit. <b><i>C</i></b>, <b><i>D</i></b>, Electron micrographs of transverse sections at the corpus callosum from mice at postnatal day 10 (C), and 3 months old (D). Scale bars, 2 µm. Percentages of myelinated axons in total axons are shown at the lower position of each panel. Data are the mean ± SEM (<i>n</i> = 4 for each group). *<i>p</i><0.05 (Student's <i>t</i>-test).</p

Early onset of MBP expression in the brain of <i>Ptprz</i>-deficient mice.

<p><b><i>A</i></b>, Schematic drawing of postulated signaling mechanisms of Ptprz and Fyn in oligodendrocyte differentiation and myelination. Fyn and Ptprz may also act on yet unidentified substrates other than p190RhoGAP to regulate the differentiation. The red arrow shows activation, whereas the blunt blue arrows represent inhibition. <b><i>B</i></b>, <b><i>C</i></b>, Western blot analyses of MBP expression in the cerebral cortex of mice at postnatal day 10 (B), and 3 months old (C). Applied protein amounts were verified by Coomassie Brilliant Blue (CBB) staining. The amounts of MBP are presented as densitometric units normalized to the value for respective wild-type controls, and are shown at the lower position of each panel. Data are the mean ± SEM (<i>n</i> = 6 for each group). **<i>p</i><0.01 (Student's <i>t</i>-test). a.u., arbitrary unit.</p

Reduced clinical severity of EAE in <i>Ptprz</i>-deficient mice.

<p>Clinical scores in wild-type mice and <i>Ptprz</i>-deficient mice after the MOG peptide injection. Clinical scores are 0, no disease; 1, limp tail; 2, ataxia and/or paresis of hindlimbs; 3, paralysis of hindlimbs and/or paresis of forelimbs; 4, tetraparalysis; 5, moribund or death. Data are the mean ± SEM (<i>Ptprz</i>^+/+, <i>n</i> = 25; <i>Ptprz</i>^−/−, <i>n</i> = 23). The comparison of clinical scores between the two groups at each time point was performed with Mann-Whitney's <i>U</i>-test, *<i>p</i><0.05, **<i>p</i><0.01.</p

Reduced tissue damage and increased oligodendrocyte survival in <i>Ptprz</i>-deficient mice with EAE.

<p><b><i>A</i></b>, <b><i>B</i></b>, Klüver-Barrera (A) and Bielschowsky silver staining (B) of the spinal cord obtained from wild-type and <i>Ptprz</i>-deficient mice 28 days after MOG immunization. The lower images are enlargements of the areas enclosed by squares in the upper images, respectively. The extent of demyelination and axon injury determined by Klüver-Barrera staining and Bielschowsky silver staining is shown as the percentage of damaged areas at the right of each panel. Scale bars, 500 µm. Data are the mean ± SEM (<i>n</i> = 10 for each group). *<i>p</i><0.05 (Student's <i>t</i>-test). <b><i>C</i></b>, TUNEL staining of spinal cord sections 35 days after MOG immunization, or non-immunized control animals. Scale bars, 100 µm. TUNEL-positive cells were counted in six sections from each animal, and the numbers of TUNEL-positive cells per section are shown at the right. Data are the mean ± SEM (<i>n</i> = 6 for each group). *<i>p</i><0.05 (Student's <i>t</i>-test).</p