Engagement of high-affinity immunoglobulin E receptors (FceRI) activates two signaling pathways i... more Engagement of high-affinity immunoglobulin E receptors (FceRI) activates two signaling pathways in mast cells. The Lyn pathway leads to recruitment of Syk and to calcium mobilization whereas the Fyn pathway leads to phosphatidylinositol 3kinase recruitment. Mapping the connections between both pathways remains an important task to be completed. We previously reported that Phospholipid Scramblase 1 (PLSCR1) is phosphorylated on tyrosine after cross-linking FceRI on RBL-2H3 rat mast cells, amplifies mast cell degranulation, and is associated with both Lyn and Syk tyrosine kinases. Here, analysis of the pathway leading to PLSCR1 tyrosine phosphorylation reveals that it depends on the FcRc chain. FceRI aggregation in Fyn-deficient mouse bone marrow-derived mast cells (BMMC) induced a more robust increase in FceRI-dependent tyrosine phosphorylation of PLSCR1 compared to wild-type cells, whereas PLSCR1 phosphorylation was abolished in Lyn-deficient BMMC. Lyn association with PLSCR1 was not altered in Fyn-deficient BMMC. PLSCR1 phosphorylation was also dependent on the kinase Syk and significantly, but partially, dependent on detectable calcium mobilization. Thus, the Lyn/Syk/calcium axis promotes PLSCR1 phosphorylation in multiple ways. Conversely, the Fyn-dependent pathway negatively regulates it. This study reveals a complex regulation for PLSCR1 tyrosine phosphorylation in FceRI-activated mast cells and that PLSCR1 sits at a crossroads between Lyn and Fyn pathways.
The cytoplasmic region of the γ chain of the high-affinity receptor for IgE (FcεRI) contains a co... more The cytoplasmic region of the γ chain of the high-affinity receptor for IgE (FcεRI) contains a consensus sequence termed the immunoreceptor tyrosine-based activation motif (ITAM). Phosphorylation of the two tyrosine residues (N-terminal Y47 and C-terminal Y58) in the ITAM sequence is crucial for the recruitment and activation of Syk, a cytoplasmic tyrosine kinase with central signaling roles in mast cells. Using a reconstitution system in which individual tyrosine-to-phenylalanine substituted γ chains were expressed in γ-chain-deficient mast cells, we previously reported differential dephosphorylation of these tyrosines. Herein, we developed monoclonal antibodies highly specific to the phosphorylated Y47 and Y58 residues, which enables monitoring their phosphorylation under more physiological conditions. Using these antibodies, preferential dephosphorylation of Y58 following FcεRI stimulation was confirmed. Furthermore, Y58 is potentially more susceptible to phosphorylation than is ...
Mycoheterotrophic orchids depend completely on mycorrhizal fungi for their supply of carbon. The ... more Mycoheterotrophic orchids depend completely on mycorrhizal fungi for their supply of carbon. The life-history traits of mycoheterotrophic plants (MHPs) can differ according to the characteristics of the associated mycorrhizal fungi. We compared the life-history strategies of two mycoheterotrophic orchids associated with wood- and leaf litter-decaying fungi over a maximum of six years of field monitoring. Seventy percent of the aboveground stems of Erythrorchis altissima, associated with wood-decaying fungi, disappeared from the host wood within two years after tagging, likely due to nutrient depletion. In contrast, Gastrodia confusa, associated with leaf litter-decaying fungi, occurred continuously (18 to 108 fruiting stalks) every year within a small-scale plot (12 × 45 m) for six years through seed and clonal propagation. Our results support the idea that mycoheterotrophic orchids associated with wood-decaying fungi disappear from their habitats due to nutrient depletion after the...
PREMISE Orchids depend primarily on mycorrhizal fungi to obtain nutrients throughout their life c... more PREMISE Orchids depend primarily on mycorrhizal fungi to obtain nutrients throughout their life cycle. Epiphytic orchids account for 69% of orchid diversity. The unstable availability of water and nutrients in their arboreal habitats often results in severe water and nutrient stresses. Consequently, mycorrhizal associations may be important for the survival of epiphytic orchids, but our understanding thereof remains limited. Here, we investigated the mycorrhizal community in a single epiphytic orchid species, using more samples than in any previous study. METHODS We assessed the mycorrhizal communities of Thrixspermum japonicum, one of the most common epiphytic orchids in the temperate region of Japan. In total, 144 individuals were collected from 28 host tree species at 20 sites across 1300 km. The mycorrhizal fungi were identified based on nuclear ribosomal DNA internal transcribed spacer sequences and assigned operational taxonomic units (OTUs) based on 97% sequence similarity. RESULTS We obtained 24 OTUs; 9 belonged to the Ceratobasidiaceae and 15 to the Tulasnellaceae. These OTUs are widely distributed throughout the phylogenetic trees of the two fungal families. However, a single Ceratobasidiaceae OTU accounted for 49.7% of all fungal sequences and was predominant in samples from 15 host tree species and 12 sites. CONCLUSIONS Our results imply that despite having a broad range of mycorrhizal partners, T. japonicum was predominantly associated with a single fungal taxon at most of the sites among the host-tree species investigated. These findings contribute to elucidating mycorrhizal symbiosis in epiphytic habitats.
Japanese Journal of Applied Entomology and Zoology
Flies infesting orchid flowers and fruits were collected from 16 orchid species from nine prefect... more Flies infesting orchid flowers and fruits were collected from 16 orchid species from nine prefectures in Japan. Fifteen orchid species collected from Fukushima to Kumamoto Prefectures were infested by Japanagromyza tokunagai Sasakawa. These results suggest that this agromyzid fly feeds on a wide range of orchid species, and is widely distributed in Japan. On the other hand, two orchids were injured by Chyliza vittata Meigen. Because these orchids were collected from Hokkaido Prefecture or high-altitude areas in Yamanashi Prefecture, this fly species seems to be distributed in cool temperate areas, but further investigations are required.
Leafless epiphytes in the Orchidaceae undergo a morphological metamorphosis in which the root has... more Leafless epiphytes in the Orchidaceae undergo a morphological metamorphosis in which the root has chloroplast-containing cortical cells and is the sole photosynthetic organ for carbon gain. All orchids are entirely dependent on mycorrhizal fungi for their carbon supply during seed germination, and this mycorrhizal association generally persists in adult plants. However, our knowledge of the mycorrhizal association of leafless epiphytic orchids remains limited, and the contribution of the mycorrhizal association to nutrient acquisition in these orchid species is largely unknown. In this study, the mycorrhizal fungi of a leafless epiphytic orchid, Taeniophyllum glandulosum, were identified molecularly using 68 mature plants and 17 seedlings. In total, 187 fungal internal transcribed spacer sequences were obtained, of which 99% were identified as Ceratobasidiaceae. These sequences were classified into five operational taxonomic units (OTUs) based on 97% sequence similarity. The most frequent sequence was OTU1, which accounted for 91% of all Ceratobasidiaceae sequences, although other phylogenetically distinct Ceratobasidiaceae fungi were detected. These results show that T. glandulosum is specifically associated with a particular group of Ceratobasidiaceae. All mycorrhizal fungi found in T. glandulosum seedlings belonged to OTU1, which was also found in adult plants on the same host tree. The mycorrhizal fungi from 13 host tree species were compared, and T. glandulosum was preferentially associated with OTU1 on 11 tree species. In conclusion, T. glandulosum is specifically associated with Ceratobasidiaceae fungi and this specific association remains throughout the orchid life cycle and is found on divergent host tree species.
Mycoheterotrophic plants are one of the most difficult plant groups to conserve because they are ... more Mycoheterotrophic plants are one of the most difficult plant groups to conserve because they are entirely dependent on symbiotic fungi. Establishment of viable culture systems would greatly aid their conservation. We describe a simple culture system for the mycoheterotrophic orchid, Gastrodia pubilabiata, that does not require laboratory facilities. The orchid is symbiotic with leaf-litter-decomposing fungi. Gastrodia pubilabiata seeds were incubated in plastic boxes or glass bottles filled with leaf litter collected from the natural habitat of the species. Seed germination was observed after 35 days and seedling development followed. Fungal isolates from seedlings were identified as Mycenaceae (Basidiomycota), a leaf-litter-decomposing mycorrhizal fungus of Gastrodia species. Our method can be used to conserve endangered mycoheterotrophic plants associated with leaf litter-decomposing fungi efficiently, and can also serve as a model system for physiological and molecular studies of...
Twenty-six microsatellite markers were developed for the endangered orchid Cypripedium japonicum ... more Twenty-six microsatellite markers were developed for the endangered orchid Cypripedium japonicum (Orchidaceae) to estimate the clonal diversity and genetic structure of the remaining populations in Japan. Microsatellite loci of C. japonicum were isolated using Ion Personal Genome Machine (PGM) sequencing. The primer sets were tested on 55 ramets sampled from two populations in Japan. Sixteen loci showed polymorphism in at least one population, with two to five alleles per locus. Observed and expected heterozygosities for the two populations ranged from 0.00 to 0.92 and 0.00 to 0.71, respectively. The microsatellite markers developed here provide a useful tool to analyze clonal structure and sexual regeneration status and will help to manage the remaining genetic variation within C. japonicum.
Journal of immunology (Baltimore, Md. : 1950), Jan 15, 1998
In this study, we demonstrate potent regulatory function of the murine killer cell inhibitory rec... more In this study, we demonstrate potent regulatory function of the murine killer cell inhibitory receptor-like molecules, paired Ig-like receptors (PIRs) or p91, using chimeric receptors expressed on the rat basophilic leukemia cell line RBL-2H3. One of the chimeras, which has the transmembrane and cytoplasmic domain of PIR-B fused to the extracellular portion of type IIB receptor for IgG, was able to inhibit the type I receptor for IgE-mediated degranulation response upon coaggregation. This chimera also suppressed cytoplasmic Ca2+ mobilization in the presence and absence of calcium ion in the extracellular medium. Tyrosine to phenylalanine point mutations at the third and fourth immunoreceptor tyrosine-based inhibitory motif-like sequences of PIR-B attenuated the inhibitory effects on degranulation and on cytoplasmic Ca2+ mobilization, indicating the important role of these tyrosines for the delivery of negative signal. In contrast, the cross-linking of another chimeric receptor comp...
Two BamHI Y and W fragments in the unique long sequence (U L) of the herpes simplex virus type 2 ... more Two BamHI Y and W fragments in the unique long sequence (U L) of the herpes simplex virus type 2 (HSV-2) were found to be heterogeneous in size among clones derived from a single strain as well as from epidemiologically unrelated isolates. More detailed restriction maps of these BamHI fragments were constructed and the heterogeneous subfragments were defined, cloned, and sequenced in order to investigate the mechanism causing the size difference. The subfragment of BamHI Y contained a tandem repeat sequence consisting of different numbers of 15 bp, 5%AGGGGCGGCTGGGGC3% as one unit among three isolates, and the subfragment of BamHI W contained the other tandem repeat sequence, 9 bp, 5%CCTCCCGCC3%. In the U L of the HSV-2 strain, these tandem repeat sequences were conserved and each repeat number appeared to be highly variable through viral genome replication. These results showed that the fragment length polymorphisms in these regions were attributable to the variation of unit numbers of the tandem repeat sequences.
Recombinant human interferon alpha 2a as well as natural human interferons alpha and beta signifi... more Recombinant human interferon alpha 2a as well as natural human interferons alpha and beta significantly suppressed the production of hepatitis B surface antigen by PLC/PRF/5 cells (which have been established from a human primary hepatocellular carcinoma and proven to carry the hepatitis B virus DNA) and inhibited proliferation of these cells in vitro. However, the production of alpha-fetoprotein by PLC/PRF/5 cells was less significantly affected by any of the interferons. These results suggest that these interferons not only suppress cellular proliferation but also selectively inhibit the action of the HBV gene which is persistently present in these cells.
Adenovirus (Ad) type 40 and 41 DNAs were directly extracted from stool specimens of children with... more Adenovirus (Ad) type 40 and 41 DNAs were directly extracted from stool specimens of children with gastroenteritis. Two new strains of Ad41, Sanekata and Ehime strain, were cloned and their restriction maps were constructed. The left terminal end of the cloned Ad41 genome, EcoRI-E fragment of the Sanekata strain and EcoRI-F fragment of the Ehime strain, had transforming ability in rat 3Y1 cells. Only one of the 35 isolates of Ad40 tested showed a different restriction profile, while three different restriction profiles were found in DNAs from Ad41 isolates.
Engagement of high-affinity immunoglobulin E receptors (FceRI) activates two signaling pathways i... more Engagement of high-affinity immunoglobulin E receptors (FceRI) activates two signaling pathways in mast cells. The Lyn pathway leads to recruitment of Syk and to calcium mobilization whereas the Fyn pathway leads to phosphatidylinositol 3kinase recruitment. Mapping the connections between both pathways remains an important task to be completed. We previously reported that Phospholipid Scramblase 1 (PLSCR1) is phosphorylated on tyrosine after cross-linking FceRI on RBL-2H3 rat mast cells, amplifies mast cell degranulation, and is associated with both Lyn and Syk tyrosine kinases. Here, analysis of the pathway leading to PLSCR1 tyrosine phosphorylation reveals that it depends on the FcRc chain. FceRI aggregation in Fyn-deficient mouse bone marrow-derived mast cells (BMMC) induced a more robust increase in FceRI-dependent tyrosine phosphorylation of PLSCR1 compared to wild-type cells, whereas PLSCR1 phosphorylation was abolished in Lyn-deficient BMMC. Lyn association with PLSCR1 was not altered in Fyn-deficient BMMC. PLSCR1 phosphorylation was also dependent on the kinase Syk and significantly, but partially, dependent on detectable calcium mobilization. Thus, the Lyn/Syk/calcium axis promotes PLSCR1 phosphorylation in multiple ways. Conversely, the Fyn-dependent pathway negatively regulates it. This study reveals a complex regulation for PLSCR1 tyrosine phosphorylation in FceRI-activated mast cells and that PLSCR1 sits at a crossroads between Lyn and Fyn pathways.
The cytoplasmic region of the γ chain of the high-affinity receptor for IgE (FcεRI) contains a co... more The cytoplasmic region of the γ chain of the high-affinity receptor for IgE (FcεRI) contains a consensus sequence termed the immunoreceptor tyrosine-based activation motif (ITAM). Phosphorylation of the two tyrosine residues (N-terminal Y47 and C-terminal Y58) in the ITAM sequence is crucial for the recruitment and activation of Syk, a cytoplasmic tyrosine kinase with central signaling roles in mast cells. Using a reconstitution system in which individual tyrosine-to-phenylalanine substituted γ chains were expressed in γ-chain-deficient mast cells, we previously reported differential dephosphorylation of these tyrosines. Herein, we developed monoclonal antibodies highly specific to the phosphorylated Y47 and Y58 residues, which enables monitoring their phosphorylation under more physiological conditions. Using these antibodies, preferential dephosphorylation of Y58 following FcεRI stimulation was confirmed. Furthermore, Y58 is potentially more susceptible to phosphorylation than is ...
Mycoheterotrophic orchids depend completely on mycorrhizal fungi for their supply of carbon. The ... more Mycoheterotrophic orchids depend completely on mycorrhizal fungi for their supply of carbon. The life-history traits of mycoheterotrophic plants (MHPs) can differ according to the characteristics of the associated mycorrhizal fungi. We compared the life-history strategies of two mycoheterotrophic orchids associated with wood- and leaf litter-decaying fungi over a maximum of six years of field monitoring. Seventy percent of the aboveground stems of Erythrorchis altissima, associated with wood-decaying fungi, disappeared from the host wood within two years after tagging, likely due to nutrient depletion. In contrast, Gastrodia confusa, associated with leaf litter-decaying fungi, occurred continuously (18 to 108 fruiting stalks) every year within a small-scale plot (12 × 45 m) for six years through seed and clonal propagation. Our results support the idea that mycoheterotrophic orchids associated with wood-decaying fungi disappear from their habitats due to nutrient depletion after the...
PREMISE Orchids depend primarily on mycorrhizal fungi to obtain nutrients throughout their life c... more PREMISE Orchids depend primarily on mycorrhizal fungi to obtain nutrients throughout their life cycle. Epiphytic orchids account for 69% of orchid diversity. The unstable availability of water and nutrients in their arboreal habitats often results in severe water and nutrient stresses. Consequently, mycorrhizal associations may be important for the survival of epiphytic orchids, but our understanding thereof remains limited. Here, we investigated the mycorrhizal community in a single epiphytic orchid species, using more samples than in any previous study. METHODS We assessed the mycorrhizal communities of Thrixspermum japonicum, one of the most common epiphytic orchids in the temperate region of Japan. In total, 144 individuals were collected from 28 host tree species at 20 sites across 1300 km. The mycorrhizal fungi were identified based on nuclear ribosomal DNA internal transcribed spacer sequences and assigned operational taxonomic units (OTUs) based on 97% sequence similarity. RESULTS We obtained 24 OTUs; 9 belonged to the Ceratobasidiaceae and 15 to the Tulasnellaceae. These OTUs are widely distributed throughout the phylogenetic trees of the two fungal families. However, a single Ceratobasidiaceae OTU accounted for 49.7% of all fungal sequences and was predominant in samples from 15 host tree species and 12 sites. CONCLUSIONS Our results imply that despite having a broad range of mycorrhizal partners, T. japonicum was predominantly associated with a single fungal taxon at most of the sites among the host-tree species investigated. These findings contribute to elucidating mycorrhizal symbiosis in epiphytic habitats.
Japanese Journal of Applied Entomology and Zoology
Flies infesting orchid flowers and fruits were collected from 16 orchid species from nine prefect... more Flies infesting orchid flowers and fruits were collected from 16 orchid species from nine prefectures in Japan. Fifteen orchid species collected from Fukushima to Kumamoto Prefectures were infested by Japanagromyza tokunagai Sasakawa. These results suggest that this agromyzid fly feeds on a wide range of orchid species, and is widely distributed in Japan. On the other hand, two orchids were injured by Chyliza vittata Meigen. Because these orchids were collected from Hokkaido Prefecture or high-altitude areas in Yamanashi Prefecture, this fly species seems to be distributed in cool temperate areas, but further investigations are required.
Leafless epiphytes in the Orchidaceae undergo a morphological metamorphosis in which the root has... more Leafless epiphytes in the Orchidaceae undergo a morphological metamorphosis in which the root has chloroplast-containing cortical cells and is the sole photosynthetic organ for carbon gain. All orchids are entirely dependent on mycorrhizal fungi for their carbon supply during seed germination, and this mycorrhizal association generally persists in adult plants. However, our knowledge of the mycorrhizal association of leafless epiphytic orchids remains limited, and the contribution of the mycorrhizal association to nutrient acquisition in these orchid species is largely unknown. In this study, the mycorrhizal fungi of a leafless epiphytic orchid, Taeniophyllum glandulosum, were identified molecularly using 68 mature plants and 17 seedlings. In total, 187 fungal internal transcribed spacer sequences were obtained, of which 99% were identified as Ceratobasidiaceae. These sequences were classified into five operational taxonomic units (OTUs) based on 97% sequence similarity. The most frequent sequence was OTU1, which accounted for 91% of all Ceratobasidiaceae sequences, although other phylogenetically distinct Ceratobasidiaceae fungi were detected. These results show that T. glandulosum is specifically associated with a particular group of Ceratobasidiaceae. All mycorrhizal fungi found in T. glandulosum seedlings belonged to OTU1, which was also found in adult plants on the same host tree. The mycorrhizal fungi from 13 host tree species were compared, and T. glandulosum was preferentially associated with OTU1 on 11 tree species. In conclusion, T. glandulosum is specifically associated with Ceratobasidiaceae fungi and this specific association remains throughout the orchid life cycle and is found on divergent host tree species.
Mycoheterotrophic plants are one of the most difficult plant groups to conserve because they are ... more Mycoheterotrophic plants are one of the most difficult plant groups to conserve because they are entirely dependent on symbiotic fungi. Establishment of viable culture systems would greatly aid their conservation. We describe a simple culture system for the mycoheterotrophic orchid, Gastrodia pubilabiata, that does not require laboratory facilities. The orchid is symbiotic with leaf-litter-decomposing fungi. Gastrodia pubilabiata seeds were incubated in plastic boxes or glass bottles filled with leaf litter collected from the natural habitat of the species. Seed germination was observed after 35 days and seedling development followed. Fungal isolates from seedlings were identified as Mycenaceae (Basidiomycota), a leaf-litter-decomposing mycorrhizal fungus of Gastrodia species. Our method can be used to conserve endangered mycoheterotrophic plants associated with leaf litter-decomposing fungi efficiently, and can also serve as a model system for physiological and molecular studies of...
Twenty-six microsatellite markers were developed for the endangered orchid Cypripedium japonicum ... more Twenty-six microsatellite markers were developed for the endangered orchid Cypripedium japonicum (Orchidaceae) to estimate the clonal diversity and genetic structure of the remaining populations in Japan. Microsatellite loci of C. japonicum were isolated using Ion Personal Genome Machine (PGM) sequencing. The primer sets were tested on 55 ramets sampled from two populations in Japan. Sixteen loci showed polymorphism in at least one population, with two to five alleles per locus. Observed and expected heterozygosities for the two populations ranged from 0.00 to 0.92 and 0.00 to 0.71, respectively. The microsatellite markers developed here provide a useful tool to analyze clonal structure and sexual regeneration status and will help to manage the remaining genetic variation within C. japonicum.
Journal of immunology (Baltimore, Md. : 1950), Jan 15, 1998
In this study, we demonstrate potent regulatory function of the murine killer cell inhibitory rec... more In this study, we demonstrate potent regulatory function of the murine killer cell inhibitory receptor-like molecules, paired Ig-like receptors (PIRs) or p91, using chimeric receptors expressed on the rat basophilic leukemia cell line RBL-2H3. One of the chimeras, which has the transmembrane and cytoplasmic domain of PIR-B fused to the extracellular portion of type IIB receptor for IgG, was able to inhibit the type I receptor for IgE-mediated degranulation response upon coaggregation. This chimera also suppressed cytoplasmic Ca2+ mobilization in the presence and absence of calcium ion in the extracellular medium. Tyrosine to phenylalanine point mutations at the third and fourth immunoreceptor tyrosine-based inhibitory motif-like sequences of PIR-B attenuated the inhibitory effects on degranulation and on cytoplasmic Ca2+ mobilization, indicating the important role of these tyrosines for the delivery of negative signal. In contrast, the cross-linking of another chimeric receptor comp...
Two BamHI Y and W fragments in the unique long sequence (U L) of the herpes simplex virus type 2 ... more Two BamHI Y and W fragments in the unique long sequence (U L) of the herpes simplex virus type 2 (HSV-2) were found to be heterogeneous in size among clones derived from a single strain as well as from epidemiologically unrelated isolates. More detailed restriction maps of these BamHI fragments were constructed and the heterogeneous subfragments were defined, cloned, and sequenced in order to investigate the mechanism causing the size difference. The subfragment of BamHI Y contained a tandem repeat sequence consisting of different numbers of 15 bp, 5%AGGGGCGGCTGGGGC3% as one unit among three isolates, and the subfragment of BamHI W contained the other tandem repeat sequence, 9 bp, 5%CCTCCCGCC3%. In the U L of the HSV-2 strain, these tandem repeat sequences were conserved and each repeat number appeared to be highly variable through viral genome replication. These results showed that the fragment length polymorphisms in these regions were attributable to the variation of unit numbers of the tandem repeat sequences.
Recombinant human interferon alpha 2a as well as natural human interferons alpha and beta signifi... more Recombinant human interferon alpha 2a as well as natural human interferons alpha and beta significantly suppressed the production of hepatitis B surface antigen by PLC/PRF/5 cells (which have been established from a human primary hepatocellular carcinoma and proven to carry the hepatitis B virus DNA) and inhibited proliferation of these cells in vitro. However, the production of alpha-fetoprotein by PLC/PRF/5 cells was less significantly affected by any of the interferons. These results suggest that these interferons not only suppress cellular proliferation but also selectively inhibit the action of the HBV gene which is persistently present in these cells.
Adenovirus (Ad) type 40 and 41 DNAs were directly extracted from stool specimens of children with... more Adenovirus (Ad) type 40 and 41 DNAs were directly extracted from stool specimens of children with gastroenteritis. Two new strains of Ad41, Sanekata and Ehime strain, were cloned and their restriction maps were constructed. The left terminal end of the cloned Ad41 genome, EcoRI-E fragment of the Sanekata strain and EcoRI-F fragment of the Ehime strain, had transforming ability in rat 3Y1 cells. Only one of the 35 isolates of Ad40 tested showed a different restriction profile, while three different restriction profiles were found in DNAs from Ad41 isolates.
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Papers by Yumi Yamashita