bluuebunny/crossref_metadata_2025_split huggingface dataset:
Requirement of Runx1/AML1/PEBP2αB for the generation of haematopoietic cells from endothelial cells
Authors: Yokomizo Tomomasa , Ogawa Minetaro , Osato Motomi , Kanno Tomohiko , Yoshida Hisahiro , Fujimoto Tetsuhiro , Fraser Stuart , Nishikawa Satomi , Okada Hitoshi , Satake Masanobu , Noda Tetsuo , Nishikawa Shin‐Ichi , Ito Yoshiaki 
Published: March 2003

Recent studies revealing that endothelial cells derived from E8.5‐E10.5 mouse embryos give rise to haematopoietic cells appear to correspond to previous histological observations that haematopoietic cell clusters are attached to the ventral aspect of dorsal aorta in such a way as if they were budding from the endothelial cell layer. Gene disruption studies have revealed that Runx1/AML1 is required for definitive haematopoiesis but not for primitive haematopoiesis, but the precise stage of gene function is not yet known.We found that mice deficient in Runx1/AML1 (an α subunit of the transcription factor PEBP2/CBF) lack c‐Kit+ haematopoietic cell clusters in the dorsal aorta, omphalomesenteric and umbilical arteries, as well as yolk sac vessels. Moreover, endothelial cells sorted from the embryo proper and the yolk sac of AML1–/– embryos are unable to differentiate into haematopoietic cells on OP9 stromal cells, whereas colonies of AML1–/– endothelial cells can be formed in culture.These results strongly suggest that the emergence of haematopoietic cells from endothelial cells represents a major pathway of definitive haematopoiesis and is an event that also occurs in the yolk sac in vivo, as suggested by earlier in vitro experiments.
Sequence‐specific termination by T7 RNA polymerase requires formation of paused conformation prior to the point of RNA release
Authors: Song Hoseok , Kang Changwon 
Published: March 2003

BackgroundThe sequence‐specific, hairpin‐independent termination signal for the bacteriophage RNA polymerases in Escherichia coli rrnB t1 terminator consists of two modules. The upstream module includes the conserved sequence and the downstream one is U‐rich.ResultsElongation complexes of T7 RNA polymerase paused 2 bp before reaching the termination site at a 500 µm concentration of NTP. At 5–50 µm NTP, however, they paused and terminated there or resumed elongation beyond the termination site. Only at higher concentrations of NTP (500 µm), the pause complex proceeded slowly to and became incompetent at the termination site. At 4 bp or more before the termination site, the unprotected single‐stranded region of transcription bubble shrank at the trailing edge to 4–5 bp from ≈10 bp, resulting from duplex formation of the conserved sequence. The pause and bubble collapse were not observed with an inactive mutant of the termination signal.ConclusionSequence‐specific termination requires the slow elongation mode of paused conformation, working only at high concentrations of NTP for a few bp prior to the RNA release site. The collapse of bubble that was observed several base pairs before the termination site and/or the resulting duplex might subsequently lead to the paused conformation of T7 elongation complexes.
Developmental roles and molecular characterization of a Drosophila homologue of Arabidopsis Argonaute1, the founder of a novel gene superfamily
Authors: Kataoka Youhei , Takeichi Masatoshi , Uemura Tadashi 
Published: March 2003

Background Arabidopsis Argonaute1 (AGO1) is the founder of a novel gene superfamily that is conserved from fission yeasts to humans. AGO1, and several other members of this superfamily are necessary for stem cell renewal or RNA interference. However, little has been reported about their roles in animal development or about the molecular activities of any of the members.ResultsWe have isolated a Drosophila homologue of AGO1, dAGO1, in our attempt to search genetically for regulators of Wingless (Wg) signal transduction. dAGO1 is broadly expressed in the embryo and the imaginal disc. dAGO1 over‐expression at wing margins suggested that it behaves as a positive regulator in the genetic background employed. Loss‐of‐function mutations of dAGO1, unexpectedly, did not give typical segment polarity phenotypes of the wg class; instead, dAGO1 maternal and zygotic mutant embryos showed developmental defects, with malformation of the nervous system being the most prominent. The mutant decreased in the numbers of several types of neurones and glia examined. The dAGO1 protein was distributed in the cytoplasm and co‐sedimented with poly(U)‐ or poly(A)‐conjugated beads.ConclusionOur results suggest that the dAGO1 protein exerts its developmental functions by binding to RNA either directly or indirectly.
Werner helicase relocates into nuclear foci in response to DNA damaging agents and co‐localizes with RPA and Rad51
Authors: Sakamoto Shuichi , Nishikawa Kaori , Heo Seok‐Jin , Goto Makoto , Furuichi Yasuhiro , Shimamoto Akira 
Published: March 2003

BackgroundWerner syndrome (WS) is an autosomal recessive disorder with many features of premature ageing. Cells derived from WS patients show genomic instability, aberrations in the S‐phase and sensitivity to genotoxic agents. The gene responsible for WS (WRN) encodes a DNA helicase belonging to the RecQ helicase family. Although biochemical studies showed that the gene product of WRN (WRNp) interacts with proteins that participate in DNA metabolism, its precise biological function remains unclear.ResultsUsing immunocytochemistry, we found that WRNp forms distinct nuclear foci in response to DNA damaging agents, including camptothecin (CPT), etoposide, 4‐nitroquinolin‐N‐oxide and bleomycin. The presence of aphidicolin inhibited CPT‐induced WRNp foci strongly but not bleomycin‐induced foci. These WRNp foci overlapped with the foci of replication protein A (RPA) almost entirely and with the foci of Rad51 partially, implicating cooperative functions of these proteins in response to DNA damage. We also found that WRNp foci partially co‐localize with sites of 5‐bromo‐2′‐deoxy‐uridine incorporation.ConclusionsThese findings suggest that WRNp form nuclear foci in response to aberrant DNA structures, including DNA double‐strand breaks and stalled replication forks. We propose that WRNp takes part in the homologous recombinational repair and in the processing of stalled replication forks.
Identification and characterization of DAlk: a novel Drosophila melanogaster RTK which drives ERK activation in vivo
Authors: Lorén Christina E. , Scully Audra , Grabbe Caroline , Edeen Philip T. , Thomas John , McKeown Michael , Hunter Tony , Palmer Ruth H. 
Published: March 2003

Background
The mammalian receptor protein tyrosine kinase (RTK), Anaplastic Lymphoma Kinase (ALK), was first described as the product of the t(2;5) chromosomal translocation found in non‐Hodgkin's lymphoma. While the mechanism of ALK activation in non‐Hodgkin's lymphoma has been examined, to date, no in vivo role for this orphan insulin receptor family RTK has been described.
Results
We describe here a novel Drosophila melanogaster RTK, DAlk, which we have mapped to band 53 on the right arm of the second chromosome. Full‐length DAlk cDNA encodes a phosphoprotein of 200 kDa, which shares homology not only with mammalian ALK but also with the orphan RTK LTK. Analysis of both mammalian and Drosophila ALK reveals that the ALK family of RTKs contains a newly identified MAM domain within their extracellular domains. Like its mammalian counterpart, DAlk appears to be expressed in the developing CNS by in situ analysis. However, in addition to expression of DAlk in the Drosophila brain, careful analysis reveals an additional early role for DAlk in the developing visceral mesoderm where its expression is coincident with activated ERK.
Conclusion
In this paper we describe a Drosophila melanogaster Alk RTK which is expressed in the developing embryonic mesoderm and CNS. Our data provide evidence for the existence of a DAlk RTK pathway in Drosophila. We show that ERK participates in this pathway, and that it is activated by DAlk in vivo. Expression patterns of dALK, together with activated ERK, suggest that DAlk fulfils the criteria of the missing RTK pathway, leading to ERK activation in the developing visceral mesoderm.
Terminal deoxynucleotidyltransferase is negatively regulated by direct interaction with proliferating cell nuclear antigen
Authors: Ibe Shirou , Fujita Kiyoko , Toyomoto Tsuguri , Shimazaki Noriko , Kaneko Reo , Tanabe Akiko , Takebe Izumi , Kuroda Sachiko , Kobayashi Tamami , Toji Shingo , Tamai Katsuyuki , Yamamoto Hiroko , Koiwai Osamu 
Published: March 2003

Background The repertoires of Ig and TcR are generated by a combinatorial rearrangement of variable (V), diversity (D), and joining (J) segments (V(D)J recombination) in B‐ and T‐cells. Terminal deoxynucleotidyltransferase (TdT) adds extra nucleotides (N nucleotides) at the junctions of the gene segments to enhance the Ig and TcR genes diversity. Using an anti‐TdT antibody column, TdT has been purified as a member of a megadalton protein complex from rat thymus. The N region would be synthesized with the large protein complex.Results The cDNAs for proliferating cell nuclear antigen (PCNA) were isolated by yeast two‐hybrid screening as the gene products which directly interacted with TdT. The interaction between PCNA and TdT was confirmed by co‐immunoprecipitation, both in vitro and in vivo. TdT binds directly to a PCNA trimer, as shown by gel filtration. TdT interacts with PCNA in its DNA polymerization domain (DPD), but not in its BRCA‐1 C‐terminal (BRCT) domain. TdT activity was reduced to 17% of the maximum value by TdT/PCNA complex formation.Conclusion TdT interacts directly with PCNA through its DPD. A functional consequence of this interaction is the negative regulation of TdT activity. These findings suggest that TdT catalyses the addition of N nucleotides under the negative control of PCNA during V(D)J recombination.
The RGS domain‐containing fission yeast protein, Rgs1p, regulates pheromone signalling and is required for mating
Authors: Pereira Paulo S. , Jones Nic C. 
Published: March 2003

Background When nutritionally starved, the fission yeast Schizosaccharomyces pombe enters a cell differentiation process which leads to mating and meiosis. The Ste11 protein is a key regulator of this differentiation pathway, activating the transcription of mating and meiotic genes upon starvation.Results Here, we describe rgs1, a member of the Regulator of G‐protein Signalling (RGS) family, as a novel Ste11 target gene. rgs1 expression requires both an Ste11‐mediated nitrogen starvation signal and the pheromone‐induced activation of the Byr2/Byr1/Spk1 MAPK pathway. We show that rgs1 deletion results in a sensitivity to pheromone and in a mating defect. Δrgs1 cells initiate the mating pathway normally, undergoing sexual agglutination and G1 arrest, while inducing pheromone‐dependent transcription, but then fail to fuse with a mating partner while elongating abnormal conjugation tubes. Endogenous Rgs1 tagged with GFP localizes to the nucleus and cytoplasm, and this localization pattern is not altered during pheromone treatment. Importantly, Rgs1 function requires its C‐terminal RGS domain, as well as a central DEP domain and a novel homology domain present in its N‐terminal region (Fungal‐DR domain).Conclusions These results demonstrate that rgs1 expression requires nutritional starvation and pheromone signalling. Rgs1 negatively regulates pheromone signalling during mating, acting in a negative feedback loop that is essential for the mating process.
Mammalian homologue of E. coli ras‐like GTPase (ERA) is a possible apoptosis regulator with RNA binding activity
Authors: Akiyama Taishin , Gohda Jin , Shibata Saiko , Nomura Yukiko , Azuma Sakura , Ohmori Yoshihiro , Sugano Sumio , Arai Hiroyuki , Yamamoto Tadashi , Inoue Jun‐ichiro 
Published: March 2003

Background ERA (Escherichia coliRas‐like protein) is an E. coli GTP binding protein that is essential for proliferation. A DNA database search suggests that homologous sequences with ERA exist in various organisms including human, mouse, Drosophila, Caenorhabditis elegans and Antirrhinum majus. However, the physiological function of eukaryotic ERA‐like proteins is not known.Results We have cloned cDNAs encoding the entire coding region of a human homologue (H‐ERA) and a mouse homologue (M‐ERA) of ERA. The mammalian homologue of ERA consists of a typical GTPase/GTP‐binding domain and a putative K homology (KH) domain, which is known as an RNA binding domain. We performed transfection experiments with wild‐type H‐ERA or various H‐ERA mutants. H‐ERA possessing the amino acid substitution mutation into the GTPase domain induced apoptosis of HeLa cells, which was blocked by Bcl‐2 expression. Deletion of the C‐terminus, which contains a part of the KH domain, alleviated apoptosis by the H‐ERA mutant, suggesting the importance of this domain in the function of H‐ERA. We have also shown the RNA binding activity of H‐ERA by pull‐down experiments using RNA homopolymer immobilized on beads or recombinant H‐ERA proteins.Conclusion Our data suggest that H‐ERA plays an important role in the regulation of apoptotic signalling with its GTPase/GTP binding domain.
Nucleocytoplasmic transport of proteins and poly(A)+ RNA in reconstituted Tpr‐less nuclei in living mammalian cells
Authors: Shibata Satoshi , Matsuoka Yosuke , Yoneda Yoshihiro 
Published: March 2003

AbstractBackground: It is known that Tpr is a component of an intranuclear long filament which extends from the nuclear pore complex (NPC) into the nucleoplasm. Since the over‐expression of the full‐length of or some fragments of Tpr in living cells leads to the accumulation of poly(A)+ RNA within the nuclei, it is generally thought that a relationship exists between Tpr and the nuclear export of mRNA in mammalian cells. In contrast, the nuclear export of poly(A)+ RNA was not inhibited in a double deletion mutant of yeast Tpr homologues (Mlp1p and Mlp2p). Therefore, the precise function of Tpr remains unknown.Results: By microinjecting two types of polyclonal antibodies which are specific to Tpr into the cytoplasm of living mammalian interphase cells, we succeeded in reconstituting the Tpr‐less nuclei. In the Tpr‐less nuclei, the localization of the major components of the NPC, the nuclear import of SV40 T‐NLS substrates and the nuclear export of HIV Rev NES‐substrates were not affected. However poly(A)+ RNA accumulated in the non‐snRNP splicing factor SC35‐positive clusters, which became larger in size and fewer in number, compared with normal nuclei.Conclusion: These results indicate that Tpr plays a critical role in the intranuclear dynamics of RNA pol II transcripts, including the processing, intranuclear transport and targeting, as well as their translocation through the NPC in mammalian cells.
Involvement of NLK and Sox11 in neural induction in Xenopus development
Authors: Hyodo‐Miura Junko , Urushiyama Seiichi , Nagai Shin‐ichi , Nishita Michiru , Ueno Naoto , Shibuya Hiroshi 
Published: March 2003

AbstractBackground : The Wnt signal transduction pathway regulates various aspects of embryonal development and has been implicated in promoting cancer. Signalling by Wnts leads to the stabilization of cytosolic β‐catenin, which then associates with TCF transcription factors to regulate expression of Wnt‐target genes. The Wnt pathway is further subject to cross‐regulation at various levels by other components.Results: Recent evidence suggests that a specific MAP kinase pathway involving the MAP kinase kinase kinase TAK1 and the MAP kinase NLK counteract Wnt signalling. In particular, it has been shown that TAK1 activates NLK, which phosphorylates TCFs bound to β‐catenin. This phosphorylation down‐regulates the DNA‐binding activity of a TCF‐4/β‐catenin complex, and blocks activation of their target genes. To investigate the role of NLK in Xenopus development, we isolated xNLK, a Xenopus homologue of NLK. Our findings indicate that xNLK is expressed in neural tissues and induces the anterior‐neural marker gene, Otx‐2. Moreover, xSox11, which is induced by the expression of Chordin, co‐operates with xNLK to induce neural development. These molecules also interact in mammalian cells, and expression of a mutant of xNLK lacking kinase activity was found to suppress the induction of neural marker gene expression by xSox11.Conclusions : Our findings indicate that xNLK may play a role in neural development together with xSox11 during early Xenopus embryogenesis.
Functional domains of paired‐like homeoprotein Cart1 and the relationship between dimerization and transcription activity
Authors: Furukawa Keizo , Iioka Takashi , Morishita Mariko , Yamaguchi Akira , Shindo Hiroyuki , Namba Hiroyuki , Yamashita Shunichi , Tsukazaki Tomoo 
Published: March 2003

AbstractBackground: Cart1 encodes the paired‐like homeodomain in the central portion of the gene, and plays a crucial role in the developmental lineage of bone and cartilage, especially in head formation. However, its transactivation mechanism is still poorly understood, including the target gene. Here, we report biochemical dissections of Cart1 functional domains and a relationship between dimerization and transcription activity.Results: Deletion studies of GAL4‐fused Cart1 indicated that the transactivation domain is located in the middle portion of the C‐terminal domain, but the N‐terminal is also required for a full activation of the consensus palindromic binding site (TAATNNNATTA). Analysis of the basic amino acid residues at both ends of the homeodomain revealed that both sides act as nuclear localization signals, and are necessary for the cooperative binding to the palindromic sequence. In this study, two additional Cart1 isoforms that behave as dominant negatives were identified from rat chondrosarcoma cells. These isoforms suppressed the transcription activity of the wild‐type, despite loss of DNA binding ability, and could interact with the wild‐type in yeast. Finally, we demonstrated that wild‐type Cart1 forms a DNA‐independent homodimer in in vivo conditions, and that the transactivation of wild‐type Cart1 was suppressed by the N‐ or C‐terminal domain which was expressed in the nucleus.Conclusion: These results revealed that homodimerization through direct interaction is necessary for the potent transcription activity of Cart1.
Roles of aurora‐A kinase in mitotic entry and G2 checkpoint in mammalian cells
Authors: Marumoto Tomotoshi , Hirota Toru , Morisaki Tetsuro , Kunitoku Naoko , Zhang Dongwei , Ichikawa Yasuko , Sasayama Takashi , Kuninaka Shinji , Mimori Tatsuyuki , Tamaki Norihiko , Kimura Masashi , Okano Yukio , Saya Hideyuki 
Published: March 2003

AbstractBackground: Various mitotic events are controlled by Cdc2‐cyclin B and other mitotic kinases. Aurora/Ipl1‐related mitotic kinases were proved to play key roles in mitotic progression in diverse lower organisms. Aurora‐A is a mammalian counterpart of aurora/Ipl1‐related kinases and is thought to be a potential oncogene. However, the regulation of aurora‐A activation and the commitment of aurora‐A in the progression of G2‐M phase are largely unknown in mammalian cells.Results: We demonstrated that aurora‐A is activated depending on the activation of Cdc2‐cyclin B in mammalian cells. Since Cdc2‐cyclin B does not directly phosphorylate aurora‐A, indirect pathways such as the inhibition of PP1 by Cdc2‐cyclin B may act for the activation of aurora‐A kinase. Microinjection of anti‐aurora‐A antibodies into HeLa cells at late G2 phase caused a significant delay in mitotic entry. Furthermore, aurora‐A activation at G2‐M transition was inhibited by DNA damage, and the over‐expression of aurora‐A induced the abrogation of the DNA damage‐induced G2 checkpoint.Conclusions: Aurora‐A is activated downstream of Cdc2‐cyclin B and plays crucial roles in proper mitotic entry and G2 checkpoint control. Dysregulation of aurora‐A induces abnormal G2‐M transition in mammalian cells and may lead to chromosome instability, which results in the development and progression of malignant tumours.
Signalling mechanisms in sphingosine 1‐phosphate‐promoted mesangial cell proliferation
Authors: Katsuma Susumu , Hada Yuko , Ueda Toshihiro , Shiojima Satoshi , Hirasawa Akira , Tanoue Akito , Takagaki Kazuchika , Ohgi Tadaaki , Yano Junichi , Tsujimoto Gozoh 
Published: March 2003

AbstractBackground: The bioactive sphingolipid sphingosine 1‐phosphate (S1P) is formed by the activation of sphingosine kinase (SPHK) in diverse stimuli, such as platelet‐derived growth factor (PDGF). S1P acts not only as an extracellular mediator but also as an intracellular second messenger, resulting in the proliferation of various different types of cells. However, the signal transduction mechanism in S1P‐induced proliferation of mesangial cells is poorly known.Results: We examined the signalling mechanisms by which S1P and dihydro‐S1P (DHS1P), another S1P receptor agonist, induce mesangial cell proliferation. We first observed that exogenous S1P/DHS1P had additive effects on the PDGF‐promoted proliferation of mesangial cells. Treatment of mesangial cells with pertussis toxin almost completely inhibited S1P‐ and DHS1P‐induced, and slightly inhibited PDGF‐induced cell proliferation. Additionally, the ERK kinase inhibitor PD98059 partially blocked the proliferation of mesangial cells induced by all these ligands. N,N‐dimethylsphingosine, a competitive inhibitor of SPHK, reduced PDGF‐induced mesangial cell proliferation, whereas over‐expression of SPHK promoted it. We also revealed that PDGF induces SPHK mRNA expression and SPHK activity, suggesting that SPHK, which links the PDGF to the S1P signalling cascade, is, at least in part, involved in PDGF‐induced mesangial cell proliferation. Moreover, we found that extracellular S1P stimulates two S1P receptors, EDG3 and EDG5, which leads to cell proliferation and survival.Conclusions: The data show that S1P‐induced mesangial cell proliferation is mediated by EDG‐dependent and ‐independent signalling pathways. S1P may cooperate with PDGF to increase the proliferation of mesangial cells during pathophysiological processes.
Thrombopoietin‐induced CXC chemokines, NAP‐2 and PF4, suppress polyploidization and proplatelet formation during megakaryocyte maturation
Authors: Oda Masaaki , Kurasawa Yasuhiro , Todokoro Kazuo , Nagata Yuka 
Published: March 2003

AbstractBackground:  We previously reported that the expressions of two CXC chemokines, neutrophil activating peptide‐2 (NAP‐2) and platelet factor‐4 (PF‐4), were induced by megakaryocyte‐specific cytokine thrombopoietin (TPO) in mouse bone marrow megakaryocytes. The roles of these chemokines on megakaryocyte maturation/differentiation processes, including polyploidization and proplatelet formation (PPF) remain unresolved.Results:  NAP‐2 and PF‐4 suppressed the PPF of mature megakaryocytes freshly prepared from mouse bone marrow as well as that of the megakaryocyte progenitors, c‐Kit+CD41+ cells, isolated from mouse bone marrow and cultured with TPO. NAP‐2 and PF‐4 inhibited polyploidization of c‐Kit+CD41+ cells in the presence of TPO, and also inhibited the proliferation of c‐Kit+CD41+ cells.Conclusions:  NAP‐2 and PF‐4 produced by TPO stimulation in megakaryocytes suppress megakaryocyte maturation and proliferation as a feedback control.