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Articles by Mayumi Kubota in JoVE
JoVE Neuroscience
עכבר בתוך הרחם Electroporation: ג'ין מבוקרת transfection Spatiotemporal
Lab for Molecular Mechanisms of Thalamus Development, RIKEN Brain Science Institute
העברת גן שיטה למוח עכבר בפיתוח מתואר באמצעות שיטה ניתוחית ייחודית צורה מיוחדת של אלקטרודות. זו טכניקה ייחודית זו מאפשרת transfection של DNA פלסמיד temporally מרחבית, אשר יסייעו מדעני מוח רבים בחקר התפתחות המוח.
Other articles by Mayumi Kubota on PubMed
Co-occurrence of Arum- and Paris-type Morphologies of Arbuscular Mycorrhizae in Cucumber and Tomato
Colonization by arbuscular mycorrhizal (AM) fungi was investigated in cucumber (Cucumis sativus), tomato (Lycopersicon esculentum) and Clethra barbinervis (Ericales) grown in field-collected soil known from previous studies to generate Paris-type arbuscular mycorrhizae in C. barbinervis. Spores of Paraglomus, Acaulospora, Glomus, and Gigaspora were found in the soil. Formation of hyphal coils and arbusculate coils of Paris-type mycorrhizae and of arbuscules of Arum-type mycorrhizae in roots raised in this soil in the growth chamber were compared with the detection of DNA of AM fungi from the same root systems using Glomales-specific primers. Only Paris-type mycorrhizae with extensive arbusculate coils developed in C. barbinervis, but cucumber and tomato developed both Paris- and Arum-types in the same root systems. Glomaceae and Archaeosporaceae and/or Paraglomaceae were detected strongly in the DNA from both cucumber and tomato roots, in which Arum-type mycorrhizae were observed. In contrast, DNA of Glomaceae was detected more sparingly in C. barbinervis, in which Paris-type mycorrhizae dominated. Acaulosporaceae and Gigasporaceae were strongly detected in the DNA from both C. barbinervis and tomato, whereas they were more weakly detected in cucumber. These results indicate that the morphology of colonization is strongly influenced by the selection of fungi to colonize the host plant from among those in the soil environment.
New Anastomosis Groups, AG-T and AG-U, of Binucleate Rhizoctonia Spp. Causing Root and Stem Rot of Cut-Flower and Miniature Roses
ABSTRACT Root and stem rot of cut-flower roses (Rosa spp.) was observed in commercial glasshouse-grown roses in 10 prefectures of Japan from 1998 through 2001. Binucleate-like Rhizoctonia spp. were isolated mainly from the disease plants. In all, 670 isolates were divided into two types based on cultural appearance; 168 isolates of light brown to brown type and 502 isolates of whitish type. A hyphal anastomosis reaction using representative isolates from each type revealed that the light brown to brown type belonged to anastomosis group G (AG-G), whereas the whitish type (AG-CUT) failed to anastomose with tester strains of binucleate Rhizoctonia AG-A through AG-S. Neither isolates of AG-G nor AG-CUT anastomosed with tester strains of a previously reported unknown AG (AG-MIN) of binucleate Rhizoctonia spp. collected from miniature roses. In pathogenicity tests, randomly selected isolates of the three groups caused root and stem rot on cut-flower and miniature roses. To differentiate AG-CUT and AG-MIN from known AGs of binucleate Rhizoctonia spp., restriction fragment length polymorphism (RFLP) and sequence analyses of a ribosomal (r)DNA internal transcribed spacer (ITS) region were conducted. Among the eight restriction enzymes used, HaeIII produced DNA banding patterns for AG-CUT that differed from those of tester strains and AG-MIN. Additionally, restriction profiles of AG-MIN differed from those of all tester strains. AG-G isolates from cut-flower roses had the same RFLP pattern as the tester strains of AG-G. Based on the results of hyphal anastomosis and RFLP and sequence analysis of an rDNA-ITS region, we propose that AG-CUT be designated AG-T and AG-MIN be designated AG-U, two new AGs of binucleate Rhizoctonia spp. The phylogenetic tree based on the sequence data of the rDNA-ITS region showed that isolates of AG-MIN were in a distinct clade from other AGs, whereas isolates of AG-CUT were in the same clade as those of AG-A. More detailed phylogenetic analysis besides rDNA-ITS region might be necessary for AG classification of binucleate Rhizoctonia spp.
Clinical Characteristics and Surgical Management for Juxtafacet Cysts of the Lumbar Spine
Retrospective analysis of 10 cases of resection of symptomatic lumbar juxtafacet cysts in nine patients (mean age 65.4 years) investigated the relationship between surgical method and progression of spinal spondylolisthesis or cyst recurrence. Patient characteristics, surgical methods, and postoperative course were reviewed. The most common preoperative symptom, painful radiculopathy, occurred in all cases, followed by motor weakness in five, sensory loss in four, and intermittent claudication in four. All patients underwent bilateral total (n = 6) or partial laminectomy (n = 4), with minimal (n = 3) or no (n = 7) facetectomy. Cysts were gross totally resected in eight cases and partially resected in two. Concomitant fixation was not performed. Painful radiculopathy, motor weakness, and sensory disturbance all resolved, resulting in good or excellent outcome in all patients. Postoperative symptomatic spondylolisthesis had not been noted at mean 52.1 months postoperatively. However, new juxtafacet cysts were later detected on the contralateral side to the initial lesion in two patients. Surgical removal of juxtafacet cysts is recommended for immediate symptomatic relief. Concomitant spinal fixation to prevent progression of spinal spondylolisthesis or cyst recurrence depends on cyst size, involvement of surrounding structures, degree of preoperative spondylolisthesis, and facet joint destruction.
The Feedback Phase of Type I Interferon Induction in Dendritic Cells Requires Interferon Regulatory Factor 8
Dendritic cells (DCs) produce type I interferons (IFNs) in greater amounts than other cells, but the mechanisms remain elusive. Here we studied the role of a transcription factor, IRF8, in DC induction of type I IFNs. Upon newcastle disease virus (NDV) infection, bone marrow-derived plasmacytoid and conventional DCs induced IFN transcripts, exhibiting two-phase kinetics. The second, amplifying phase represented an IFN feedback response that accounted for much of IFN protein production. Induction of second phase transcription required IRF8. Mouse cytomegalovirus (MCMV) and Toll-like receptor-mediated IFN induction in DCs also required IRF8. Chromatin immunoprecipitation analysis showed that IRF7, IRF8, and RNA polymerase II were recruited to the IFN promoters upon stimulation. Moreover, sustained RNA polymerase II recruitment to the promoters critically depended on IRF8. Together, these data indicate that IRF8 magnifies the second phase of IFN transcription in DCs by prolonging binding of basic transcription machinery to the IFN promoters, thereby playing a role in innate immunity.
The Plant Growth-promoting Fungus Penicillium Simplicissimum GP17-2 Induces Resistance in Arabidopsis Thaliana by Activation of Multiple Defense Signals
Arabidopsis thaliana grown in soil amended with barley grain inocula of Penicillium simplicissimum GP17-2 or receiving root treatment with its culture filtrate (CF) exhibited clear resistance to Pseudomonas syringae pv. tomato DC3000 (Pst). To assess the contribution of different defense pathways, Arabidopsis genotypes implicated in salicylic acid (SA) signaling expressing the NahG transgene or carrying disruption in NPR1 (npr1), jasmonic acid (JA) signaling (jar1) and ethylene (ET) signaling (ein2) were tested. All genotypes screened were protected by GP17-2 or its CF. However, the level of protection was significantly lower in NahG and npr1 plants than it was in similarly treated wild-type plants, indicating that the SA signaling pathway makes a minor contribution to the GP17-2-mediated resistance and is insufficient for a full response. Examination of local and systemic gene expression revealed that GP17-2 and its CF modulate the expression of genes involved in both the SA and JA/ET signaling pathways. Subsequent challenge of GP17-2-colonized plants with Pst was accompanied by direct activation of SA-inducible PR-2 and PR-5 genes as well as potentiated expression of the JA-inducible Vsp gene. In contrast, CF-treated plants infected with Pst exhibited elevated expression of most defense-related genes (PR-1, PR-2, PR-5, PDF1.2 and Hel) studied. Moreover, an initial elevation of SA responses was followed by late induction of JA responses during Pst infection of induced systemic resistance (ISR)-expressing plants. In conclusion, we hypothesize the involvement of multiple defense mechanisms leading to an ISR of Arabidopsis by GP17-2.
Syringomyelia Caused by Intrathecal Remnants of Oil-based Contrast Medium
Oily contrast medium had been in use since the early 19th century as a radiographic agent for detecting spinal lesions and spinal cord tumors until the late 20th century. At that point computed tomography scanning and magnetic resonance imaging, or other hydrophilic contrast medium substituted for it. Adverse effects of oil-based dye, both acute and chronic, had been reported since the middle of the 20th century. In this paper the authors report the case of syringomyelia that seemed to be caused mainly by remaining oily contrast medium for 44 years. Syringomyelia secondary to adhesive arachnoiditis caused by oily contrast medium after a long period of time is well known. In the present case, however, surgery revealed only mild arachnoiditis at the level of syringomyelia as well as both solid and liquid remnants of contrast medium. Generally, cerebrospinal fluid (CSF) blockage due to an arachnoid adhesion is considered to cause syringomyelia following adhesive arachnoiditis. The authors speculated that in the present case syringomyelia was induced by a mechanism different from that in the previously reported cases; the oily contrast medium itself seems to have induced the functional block of CSF and impaired the buffer system of the intrathecal pressure. No reports on thoracic adhesive arachnoiditis and syringomyelia caused by oil-based dye referred to this mechanism in reviewing the literature.
Heterokaryon Formation in Thanatephorus Cucumeris (Rhizoctonia Solani) AG-1 IC
Approximately 50 single-basidiospore isolates (SBIs) obtained from each of 16 field isolates of Thanatephorus cucumeris AG-1 IC were examined for heterokaryon formation. All SBIs obtained from each field isolate were divided into two mating groups (SBIs-M1 and SBIs-M2), and tufts of mycelia were formed in the contact zone between colonies of paired SBIs-M1 and -M2 based on 0.5 % charcoal agar medium. Tufts were produced from all possible pairing between SBIs from non-parental field isolates. Hyphal anastomosis reactions indicated no cell death and random cell death at the contact cell, and was not related to tuft formation. AFLP phenotypes of SBIs from each field isolate were not identical to each other and were different from their parental field isolate. AFLP phenotypes of the tuft isolates formed from SBIs-M1 and SBIs-M2 from each field isolate were heterokaryotic. Moreover, several SBIs also formed tufts with their parental and non-parental field isolates. AFLP phenotypes of these tuft isolates suggested that they were all heterokaryotic. Results of these experiments suggest that T. cucumeris AG-1 IC is heterothallic and bipolar, and that genetic exchange can occur between homokaryotic and heterokaryotic isolates (Buller phenomenon).
High Level Expression of a Virus Resistance Gene, RCY1, Confers Extreme Resistance to Cucumber Mosaic Virus in Arabidopsis Thaliana
A coiled coil-nucleotide binding site-leucine rich repeat-type resistance gene, RCY1, confers resistance to a yellow strain of Cucumber mosaic virus, CMV(Y), in Arabidopsis thaliana ecotype C24. Resistance to CMV(Y) in C24 is accompanied by a hypersensitive response (HR) that is characterized by the development of necrotic local lesions at the primary infection sites. To further study the HR and resistance to CMV(Y) in ecotype Col-0, which is susceptible to CMV(Y), Col-0 were transformed with RCY1. Systemic spread of CMV(Y) was completely suppressed in RCY1-transformed Col-0 (Col::pRCY1 lines 2 to 6), whereas virulent strain CMV(B2) spread and multiplied systemically in these transgenic lines similar to that in wild-type Col-0. Interestingly, the resistant phenotype of Col::pRCY1 varied among the lines. In lines 3 and 6, in which levels of RCY1 transcript were similar to that in wild-type C24, the HR and resistance to CMV(Y) was induced. Line 4, which expresses moderately elevated levels of RCY1 transcript, exhibited moderately enhanced resistance compared with that in C24 or line 3. In contrast, lines 2 and 5, which highly overexpress the RCY1 gene, did not exhibit either visible lesions or a micro-HR on the inoculated leaves. Moreover, virus coat protein was not detected in either inoculated or noninoculated upper leaves of these two lines, suggesting that extreme resistance (ER) to CMV(Y) was induced by high levels of expression of RCY1. Furthermore, in transgenic lines expressing hemagglutinin (HA) epitope-tagged RCY1 (Col::pRCY1-HA), high levels of accumulation of RCY1-HA protein were also correlated with the ER phenotype. Global gene expression analysis in line 2, which highly overexpresses RCY1, indicated that expression of several defense-related genes were constitutively elevated compared with wild-type Col-0. Despite this, line 2 did not have enhanced resistance to other avirulent and virulent pathogens. Take together, constitutive accumulation of high levels of RCY1 protein appears to regulate the strength of RCY1-conferred resistance in a gene-for-gene manner and implies that ER and HR-associated resistance differ only in the strength of resistance.
Enhanced Defense Responses in Arabidopsis Induced by the Cell Wall Protein Fractions from Pythium Oligandrum Require SGT1, RAR1, NPR1 and JAR1
The cell wall protein fraction (CWP) is purified from the non-pathogenic biocontrol agent Pythium oligandrum and is composed of two glycoproteins (POD-1 and POD-2), which are structurally similar to class III elicitins. In tomato plants treated with CWP, jasmonic acid (JA)- and ethylene (ET)-dependent signaling pathways are activated, and resistance to Ralstonia solanaceraum is enhanced. To dissect CWP-induced defense mechanisms, we investigated defense gene expression and resistance to bacterial pathogens in Arabidopsis thaliana ecotype Col-0 treated with CWP. When the leaves of Col-0 were infiltrated with CWP, neither visible necrosis nor salicylic acid (SA)-responsive gene (PR-1 and PR-5) expression was induced. In contrast, JA-responsive gene (PDF1.2 and JR2) expression was up-regulated and the resistance to R. solanaceraum and Pseudomonas syringae pv. tomato DC3000 was enhanced in response to CWP. Such CWP-induced defense responses were completely compromised in CWP-treated coi1-1 and jar1-1 mutants with an impaired JA signaling pathway. The induction of defense-related gene expression after CWP treatment was partially compromised in ET-insensitive ein2-1 mutants, but not in SA signaling mutants or nahG transgenic plants. Global gene expression analysis using cDNA array also suggested that several other JA- and ET-responsive genes, but not SA-responsive genes, were up-regulated in response to CWP. Further analysis of CWP-induced defense responses using another eight mutants with impaired defense signaling pathways indicated that, interestingly, the induction of JA-responsive gene expression and enhanced resistance to two bacterial pathogens in response to CWP were completely compromised in rar1-1, rar1-21, sgt1a-1, sgt1b (edm1) and npr1-1 mutants. Thus, the CWP-induced defense system appears to be regulated by JA-mediated and SGT1-, RAR1- and NPR1-dependent signaling pathways.
