bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification and expression of a novel gene in odour‐taste associative learning in the terrestrial slug
Authors: Nakaya Tadashi , Kawahara Shigenori , Watanabe Satoshi , Lee Dong‐Seok , Suzuki Toshiharu , Kirino Yutaka 
Published: March 2003

Odour‐taste associative learning in the terrestrial slug offers a useful model for long‐term memory formation and retention. The genes which are expressed over 2 h after a learning event are thought to include those related to memory formation and/or consolidation. It is very important to examine what kinds of genes are expressed following associative learning.We identified a novel slug gene, the expression of which was regulated by associative learning and mostly restricted to the procerebrum (PC), a place that olfactory information is believed to be processed in slug. This gene encodes a 121 amino acid, 18 kDa secretory protein which we term LAPS18. Expression of the LAPS18 gene was induced in somata and the protein spread to neurites in the PC of slugs subjected to paired conditioning. Recombinant LAPS18 promoted the aggregation and movement of PC neurones in culture and they were blocked by the anti‐LAPS18 antibody. Beads coupled with LAPS18 protein attached to PC neurones and the beads aggregated through PC cells but not by themselves, suggesting that LAPS18 may require a counterpart molecule for PC neurone aggregation.An increased expression and translocation of LAPS18 protein after paired conditioning may be needed for long‐term memory formation and retention in the slug. Since genes homologous to LAPS18 genes in the land slug Limax are found from vertebrates including human, analysing the expression and function of LAPS18 may be important in understanding the molecular mechanism of memory formation and retention.
Influenza virus RNA polymerase PA subunit is a novel serine protease with Ser624 at the active site
Authors: Hara Koyu , Shiota Mayumi , Kido Hiroshi , Ohtsu Yasushi , Kashiwagi Takahito , Iwahashi Jun , Hamada Nobuyuki , Mizoue Kazutoshi , Tsumura Naoki , Kato Hirohisa , Toyoda Tetsuya 
Published: March 2003

BackgroundInfluenza virus RNA polymerase is a multifunctional enzyme that catalyses both transcription and replication of the RNA genome. The function of the influenza virus RNA polymerase PA subunit in viral replication is poorly understood, although the enzyme is known to be required for cRNA → vRNA synthesis. The protease related activity of PA has been discussed ever since protease‐inducing activity was demonstrated in transfection experiments.ResultsPA protein was highly purified from insect cells infected with the recombinant baculovirus carrying PA cDNA, and a novel chymotrypsin‐type serine protease activity was identified with the synthetic peptide, Suc‐LLVY‐MCA, in the PA protein. [3H]DFP was crosslinked with PA and a mutational analysis revealed that serine624 was as an active site for the protease activity.ConclusionsThese results constitute the demonstration of protease activity in PA subunit of the influenza virus RNA polymerase complexes.
Selenocysteine codons decrease polysome association on endogenous selenoprotein mRNAs
Authors: Martin III Glover W. , Berry Marla J. 
Published: March 2003

BackgroundSelenocysteine incorporation has been reported to be inefficient in all systems studied, including Escherichia coli, baculovirus‐insect cell systems, rabbit reticulocyte in vitro translation systems, transiently transfected mammalian cells, and intact animals. Nonetheless, full‐length selenoproteins containing up to 17 selenocysteine residues are produced in animals, indicating that the efficiency observed in manipulated systems might not accurately reflect the true efficiency of this process in nature.ResultsTo begin to address this apparent discrepancy, we have examined the polysome profiles of endogenously expressed selenoprotein mRNAs in a mammalian cell line, and compared them with nonselenoprotein mRNAs. We report that three selenoprotein mRNAs, type 1 deiodinase, glutathione peroxidase and selenoprotein P, are under‐loaded with ribosomes, based on their predicted open reading frame sizes. The average numbers of ribosomes per mRNA correspond to the sizes predicted by termination at the UGA selenocysteine codons. Appropriate loading on the type 1 deiodinase mRNA is seen following substitution of a cysteine codon for the selenocysteine codon, indicating that the UGA codon confers a translational penalty on the mRNA. Surprisingly, ribosomal loading is also increased by the expression of eukaryotic release factors eRF1 and eRF3.ConclusionsThese results suggest that the presence of a selenocysteine codon confers a translational penalty on selenoprotein mRNAs, and that increased levels of release factors may alter the kinetics of termination.
Crk family adaptor proteins trans‐activate c‐Abl kinase
Authors: Shishido Tomoyuki , Akagi Tsuyoshi , Chalmers Alistair , Maeda Makiko , Terada Toshie , Georgescu Maria‐Magdalena , Hanafusa Hidesaburo 
Published: March 2003

Backgroundc‐Abl kinase is activated in response to a variety of biological stimuli. Crk family adaptor proteins can interact physically with c‐Abl and be involved in the activation of c‐Abl kinase.ResultsWe report that the Crk family of adaptor proteins act as trans‐acting activators of c‐Abl kinase. The interaction of the amino‐terminal Src‐homology (SH) 3 domain of c‐Crk and the proline‐rich motifs of c‐Abl is an essential step for the phosphorylation of c‐Crk by c‐Abl, as well as the activation of c‐Abl by c‐Crk. The activation of c‐Abl by c‐Crk is negatively regulated by phosphorylation of the tyrosine 221 of c‐Crk. Our data suggest that, in the absence of phosphorylation of the tyrosine Y221, the SH2 domain of c‐Crk becomes free to bind to target molecules while the carboxyl‐terminal SH3 domain of c‐Crk binds to the proline‐rich region of c‐Abl, inducing the activation of c‐Abl by c‐Crk.ConclusionThis study suggests that the Crk family functions as trans‐acting activators of c‐Abl kinase. The phosphorylation of c‐Crk may regulate c‐Abl kinase.
Self‐splicing of the Tetrahymena group I ribozyme without conserved base‐triples
Authors: Ikawa Yoshiya , Yoshioka Wataru , Ohki Yasushi , Shiraishi Hideaki , Inoue Tan 
Published: March 2003

BackgroundGroup I introns share a conserved core region consisting of two domains, P8‐P3‐P7 and P4‐P6, joined by four base‐triples. We showed previously that the T4 td intron can perform phosphoester transfer reactions at two splice sites in the absence of both P4‐P6 and the conserved base‐triples, whereas it is barely able to perform the intact splicing reaction due to the difficulty of conducting the sequential reactions.ResultsBased on previous findings, we constructed a bimolecular ribozyme lacking a large portion of P4‐P6 and the base‐triples from the Tetrahymena intron, on the assumption that the long–range interactions of the peripheral regions in the two RNAs can compensate for the deteriorated core. The bimolecular ribozyme performed the intact splicing reaction.ConclusionThe present analysis indicates that the base‐triples are nonessential, but that L4 and the distal part of P4 in P4‐P6 are important for conducting the splicing reaction. The reconstituted self‐splicing ribozyme provides an amenable system for analysing the role(s) of elements in the core region in the self‐splicing reaction mechanism.
Yeast Saccharomyces cerevisiae has two cis‐prenyltransferases with different properties and localizations. Implication for their distinct physiological roles in dolichol synthesis
Authors: Sato Miyuki , Fujisaki Shingo , Sato Ken , Nishimura Yukinobu , Nakano Akihiko 
Published: March 2003

Background Dolichol is a family of long‐chain polyprenols, which is utilized as a sugar carrier in protein glycosylation in the endoplasmic reticulum (ER). We have identified a key enzyme of the dolichol synthesis, cis‐prenyltransferase, as Rer2p from Saccharomyces cerevisiae. We have also isolated a multicopy suppressor of an rer2 mutant and named it SRT1. It encodes a protein similar to Rer2p but its function has not been established.Results The cis‐prenyltransferase activity of Srt1p has been proved biochemically in the lysate of yeast cells lacking Rer2p. The polyprenol product of Srt1p is longer in chain length than that of Rer2p and is not sufficiently converted to dolichol and dolichyl phosphate, unlike that of Rer2p. The subcellular localization of these two isozymes has been examined by immunofluorescence microscopy and by the use of GFP fusion proteins. Whereas GFP‐Rer2p is localized to the continuous ER and some dots associated with the ER, GFP‐Srt1p shows only punctate localization patterns. Immunofluorescence double staining with Erg6p, a marker of lipid particles in yeast, indicates that Srt1p is mainly localized to lipid particles (lipid bodies). RER2 is mainly expressed in the early logarithmic phase, while the expression of SRT1 is induced in the stationary phase.Conclusions We have shown that yeast has two active cis‐prenyltransferases with different properties. This result implies that the two isozymes have different physiological roles during the life cycle of the yeast.
Inhibition of BMP2‐induced, TAK1 kinase‐mediated neurite outgrowth by Smad6 and Smad7
Authors: Yanagisawa Makoto , Nakashima Kinichi , Takeda Kohsuke , Ochiai Wataru , Takizawa Takumi , Ueno Masaya , Takizawa Makiko , Shibuya Hiroshi , Taga Tetsuya 
Published: March 2003

Background BMP2 is known to play a wide variety of roles, including some in the development of the nervous system. This cytokine has been reported to induce neurite outgrowth in rat pheochromocytoma PC12 cells via the activation of a p38 MAP kinase, although its regulatory mechanism remains largely to be elucidated.Results BMP2‐induced neurite outgrowth in PC12 cells was inhibited by the introduction of a kinase‐negative form of a MAP kinase kinase kinase, TAK1, an upstream regulatory kinase for p38 kinase. Following BMP2 stimulation, the expression of Smad6 and Smad7, inhibitory Smad species that are known to inhibit the BMP2‐restricted Smad species, Smad1, Smad5 and Smad8, was up‐regulated. Unexpectedly, over‐expression of either Smad6 or Smad7 in PC12 cells repressed the BMP2‐induced neurite outgrowth and severely impeded the p38 kinase pathway. Both of these inhibitory Smads were found to interact physically with TAK1‐binding protein, a molecule required for TAK1 activation.Conclusions This study demonstrates that BMP2‐induced neurite outgrowth in PC12 cells involves activation of the TAK1‐p38 kinase pathway which is inhibited by Smad6 and Smad7.
Step‐wise divergence of primitive and definitive haematopoietic and endothelial cell lineages during embryonic stem cell differentiation
Authors: Fujimoto Tetsuhiro , Ogawa Minetaro , Minegishi Naoko , Yoshida Hisahiro , Yokomizo Tomomasa , Yamamoto Masayuki , Nishikawa Shin‐ichi 
Published: March 2003

Background The developmental processes leading from the mesoderm to primitive and definitive haematopoietic and endothelial lineages, although of great importance, are still poorly defined. Recent studies have suggested a model in which common precursors give rise to endothelial progenitors and haematopoietic progenitors, the latter subsequently generating both primitive and definitive haematopoietic lineages. However, this model is contradicted by findings that suggest the emergence of haematopoietic cells from the endothelial lineage.Results We found sequential steps in the differentiation of FLK1+ mesoderm into haematopoietic and endothelial lineages in an in vitro differentiation system of embryonic stem (ES) cells: (i) the GATA‐1+ subset of FLK1+ mesodermal cells loses the capacity to give rise to endothelial cells and is restricted to primitive erythroid, macrophage and definitive erythroid progenitors; (ii) the remaining GATA‐1− cells give rise to VE‐cadherin+ endothelial cells; and subsequently (iii) multiple definitive haematopoietic progenitors and endothelial cells branch off from a subset of VE‐cadherin+ cells.Conclusions These observations strongly suggest that the divergence of primitive and multilineage definitive haematopoietic/endothelial lineages occurs first, and then multilineage definitive haematopoietic progenitors arise from VE‐cadherin+ endothelial cells in the development of haematopoietic and endothelial cells.
Characterization, expression and complex formation of the murine Fanconi anaemia gene product Fancg
Authors: Van De Vrugt Henri J. , Koomen Mireille , Berns Mariska A. D. , De Vries Yne , Rooimans Martin A. , Van Der Weel Laura , Blom Eric , De Groot Jan , Schepers Rik J. , Stone Stacie , Hoatlin Maureen E. , Cheng Ngan Ching , Joenje Hans , Arwert Fré 
Published: March 2003

AbstractBackground: Fanconi anaemia (FA) is an autosomal recessive chromosomal instability disorder. Six distinct FA disease genes have been identified, the products of which function in an integrated pathway that is thought to support a nuclear caretaker function. Comparison of FA gene characteristics in different species may help to unravel the molecular function of the FA pathway.Results: We have cloned the murine homologue of the Fanconi anaemia complementation group G gene, FANCG/XRCC9. The murine Fancg protein shows an 83% similarity to the human protein sequence, and has a predicted molecular weight of 68.5 kDa. Expression of mouse Fancg in human FA‐G lymphoblasts fully corrects their cross‐linker hypersensitivity. At mRNA and protein levels we detected the co‐expression of Fancg and Fanca in murine tissues. In addition, mouse Fancg and Fanca proteins co‐purify by immunoprecipitation. Upon transfection into Fanca‐deficient mouse embryonic fibroblasts EGFP‐Fancg chimeric protein was detectable in the nucleus.Conclusions: We identified a murine cDNA, Fancg, which cross‐complements the cellular defect of human FA‐G cells and thus represents a true homologue of human FANCG. Spleen, thymus and testis showed the highest Fancg expression levels. Although Fancg and Fanca are able to form a complex, this interaction is not required for Fancg to accumulate in the nuclear compartment.
Tropomyosin is required for the cell fusion process during conjugation in fission yeast
Authors: Kurahashi Hiroshi , Imai Yoshiyuki , Yamamoto Masayuki 
Published: March 2003

AbstractBackground:  Tropomyosin is an actin‐binding protein, which is thought to stabilize actin filaments and influence many aspects of F‐actin. In fission yeast, the cdc8 gene encodes tropomyosin, and the gene product Cdc8p is known to be essential for the formation of the F‐actin contractile ring and hence for cytokinesis in the mitotic cell cycle.Results: We isolated fission yeast mutants that were defective in cell fusion during conjugation. One of them turned out to carry a point mutation in cdc8. We found that the original temperature‐sensitive cdc8 mutant frequently failed to undergo cell fusion when mated at a semi‐permissive temperature. Additional cdc8 mutants isolated by targeted mutagenesis also showed defects in both cell fusion and cytokinesis. A decrease in the amount of intracellular Cdc8p also affected both, but cell growth was more severely blocked than cell fusion in this case. Immunostaining revealed that Cdc8p was localized as a spot at the cell‐to‐cell attachment site during conjugation, without overlapping with F‐actin patches.Conclusions: Tropomyosin Cdc8p is indispensable for cell fusion during conjugation in fission yeast. However, cell fusion appears to require fewer tropomyosin molecules than cytokinesis. We speculate that tropomyosin may organize a small F‐actin‐containing organelle at the cell‐to‐cell contact site in each mating cell, which plays a key role in cell fusion.
Hyper‐processive and slower DNA chain elongation catalysed by DNA polymerase III holoenzyme purified from the dnaE173 mutator mutant of Escherichia coli
Authors: Sugaya Yutaka , Ihara Keiichi , Masuda Yuji , Ohtsubo Eiichi , Maki Hisaji 
Published: March 2003

AbstractBackground: A strong mutator mutation, dnaE173, leads to a Glu612 → Lys amino acid change in the α subunit of Escherichia coli DNA polymerase III (PolIII) holoenzyme and abolishes the proofreading function of the replicative enzyme without affecting the 3′ → 5′ exonuclease activity of the ɛ subunit. The dnaE173 mutator is unique in its ability to induce sequence‐substitution mutations, suggesting that an unknown function of the α subunit is hampered by the dnaE173 mutation.Results: A PolIII holoenzyme reconstituted from dnaE173 PolIII* (DNA polymerase III holoenzyme lacking the β clamp subunit) and the β subunit showed a strong resistance to replication‐pausing on the template DNA and readily promoted strand‐displacement DNA synthesis. Unlike wild‐type PolIII*, dnaE173 PolIII* was able to catalyse highly processive DNA synthesis without the aid of the β‐clamp subunit. The rate of chain elongation by the dnaE173 holoenzyme was reduced to one‐third of that determined for the wild‐type enzyme. In contrast, an exonuclease‐deficient PolIII holoenzyme was vastly prone to pausing, but had the same rate of chain elongation as the wild‐type.Conclusions: The hyper‐processivity and slower DNA chain elongation rate of the dnaE173 holoenzyme are distinct effects caused by the dnaE173 mutation and are likely to be involved in the sequence‐substitution mutagenesis. A link between the proofreading and chain elongation processes was suggested.
Carboxyl‐terminal basic amino acids in the X domain are essential for the nuclear import of phospholipase C δ1
Authors: Okada Masashi , Fujii Makoto , Yamaga Masaki , Sugimoto Hiroaki , Sadano Hiroyuki , Osumi Takashi , Kamata Hideaki , Hirata Hajime , Yagisawa Hitoshi 
Published: March 2003

AbstractBackground: Although phospholipase C (PLC)δ1 containing a functional nuclear export signal (NES) is normally localized at the plasma membrane and in the cytoplasm, it shuttles between the nucleus and the cytoplasm. Since nucleocytoplasmic shuttling of a molecule is generally regulated by a balance between its NES and the nuclear localization signal (NLS), we examined whether PLCδ1 contains an NLS sequence.Results: A region corresponding to the C terminus of the X domain and the XY‐linker, which contains clusters of basic amino acid residues, was essential for the nuclear import of PLCδ1 in Madin–Darby canine kidney cells. A series of point mutations on lysine residues in this region revealed that K432 and K434 in combination were important for the nuclear import. A short synthetic peptide corresponding to residues 429–442, however, was not able to function as an NLS sequence when they were injected into the cytoplasm in a carrier‐conjugated form. Neither a longer peptide equivalent to PLCδ1 412–498 fused to a protein tag consisting of glutathione S‐transferase and green fluorescent protein was imported to the nucleus after microinjection into the cytoplasm.Conclusion: The nuclear import of PLCδ1 requires the C‐terminus of the X domain, particularly the amino acid residues K432 and K434, and the XY‐linker. The region alone, however, cannot serve as a functional NLS. The machinery for nuclear transport may require additional structural component(s) of the enzyme.
Calcineurin is implicated in the regulation of the septation initiation network in fission yeast
Authors: Lu Yabin , Sugiura Reiko , Yada Tomoko , Cheng Hong , Sio Susie O. , Shuntoh Hisato , Kuno Takayoshi 
Published: March 2003

AbstractBackground: In fission yeast, calcineurin has been implicated in cytokinesis because calcineurin‐deleted cells form multiple septa and cell separation is impeded. However, this mechanism remains unclear.Results: We screened for mutations that confer syn‐thetic lethality with calcineurin deletion and isolated a mutant, its10‐1/cdc7‐i10, a novel allele of the cdc7+ gene involved in the septation initiation network (SIN). The mutation created a termination codon, resulting in the truncation of Cdc7 by 162 amino acids, which is not localized in the spindle pole body. Following treatment with the immune suppressive drug FK506, cdc7‐i10 and the original cdc7‐24 mutant cells showed highly elongated multinuclear morphology with few visible septa, closely resembling the phenotype at the restrictive temperature. Other SIN mutants, cdc11, spg1, sid2 and mob1 showed similar phenotypes following FK506 treatment. Consistent with this, expression of the constitutively active calcineurin suppressed the growth defects and septum initiation deficiency of these SIN mutants at the restrictive temperature. Moreover, electron microscopy revealed that calcineurin‐deleted cells had very thick multiple septa which were partially and ectopically formed.Conclusion: These results suggest that calcineurin is involved in the regulation of the SIN pathway, and is required for the proper formation and maturation of the septum in fission yeast.
Isolation and expression of a novel mitochondrial septin that interacts with CRMP/CRAM in the developing neurones
Authors: Takahashi Shusuke , Inatome Ryoko , Yamamura Hirohei , Yanagi Shigeru 
Published: March 2003

AbstractBackground: Collapsin response mediator proteins (CRMPs) and CRAM belong to the unc‐33 gene family which is implicated in axon guidance and outgrowth during neural development. However, their exact roles remain largely unknown. To understand the molecular basis of CRMP/CRAM function, we have undertaken to identify CRMP/CRAM interacting proteins.Results: We have identified a novel mitochondrial septin (M‐septin) as one of the CRMP/CRAM interacting proteins from the developing rat brain. M‐septin is a major, alternatively spliced variant of the H5 gene in developing mouse brain and its expression is up‐regulated during the neuronal differentiation of embryonal carcinoma P19 cells. In COS‐7 cells, M‐septin is specifically localized to mitochondria whereas H5 is diffusely distributed to the perinuclear cytoplasm and plasma membranes. In contrast to H5, M‐septin induces the mitochondrial translocation of CRAM but not CRMP2. Finally, M‐Septin is found to be transiently translocated to mitochondria before the induction of the neurites and then dissociates from the mitochondria after neurite extension in P19 cells.Conclusions: Our results suggest that M‐septin has a role which is distinct from H5, and together with CRMP/CRAM, may play an important role in the neuronal differentiation and axon guidance through the control of mitochondrial function.
MDG1/ERdj4, an ER‐resident DnaJ family member, suppresses cell death induced by ER stress
Authors: Kurisu Junko , Honma Asuka , Miyajima Hiroshi , Kondo Shinichi , Okumura Masayo , Imaizumi Kazunori 
Published: March 2003

AbstractBackground: Alterations in homeostasis after various cellular stresses, which prevent protein folding and cause an accumulation of misfolding or malfolding proteins in the endoplasmic reticulum (ER), have the potential to induce cellular damage, and are therefore a type of ‘ER stress.’ To understand the molecular events or cascades underlying the ER stress response regulated by gene transcription and mediated by stress transducers, it is crucial to identify the molecules induced during ER stress and to analyse the roles of these genes.Results: We identified MDG1/ERdj4, a member of the DnaJ protein family, as an inducible gene during ER stress. MDG1/ERdj4 contains the J domain, which is essential for interacting with Hsp70s, at the N‐terminal portion and just at the back of the transmembrane domain. Its trypsin digestion and glycosylation of a chimeric protein composed of MDG1/ERdj4 fused with the extracellular domain of the amyloid precursor protein at its C‐terminus, showed that its C‐terminal portion containing the J domain could be orientated to the ER lumen. Over‐expression of it inhibited the cell death induced by ER stress. In contrast, its mutants with the J domain deleted showed no protective effects against cell death.Conclusions: MDG1/ERdj4 may play roles in stabilizing GRP78/BiP binding to unfolded substrate proteins in a J domain‐dependent manner and prevent the accumulation of unfolded proteins in the ER, consequently protecting cells from ER stress.