bluuebunny/crossref_metadata_2025_split huggingface dataset:
Carboxyl‐terminal region conserved among phosphoinositide‐kinase‐related kinases is indispensable for mTOR function in vivo and in vitro
Authors: Takahashi Tetsuya , Hara Kenta , Inoue Hitomi , Kawa Yoshitaka , Tokunaga Chiharu , Hidayat Sujuti , Yoshino Ken‐ichi , Kuroda Yoshikazu , Yonezawa Kazuyoshi 
Published: March 2003

BackgroundThe mammalian target of rapamycin (mTOR) belongs to the family of phosphoinositide (PI)‐kinase‐related kinases that includes the ataxia‐telangiectasia gene product (ATM). mTOR plays a critical role in controlling translational effectors such as p70 S6 kinase α (p70α) and eukaryotic initiation factor 4E binding protein 1 (4EBP1).ResultsWe show that the C‐terminal region of mTOR, which is highly conserved among the PI‐kinase‐related kinases, plays a critical role in the mTOR protein kinase activity. Deletion of the C‐terminal residues did not adversely affect the expression of mTOR, but caused a nearly complete loss of the mTOR protein kinase activity toward both 4EBP1 and p70αin vitro. These deletions also abolished the ability of a rapamycin‐resistant mTOR mutant to rescue the activity of p70α from inhibition induced by rapamycin in vivo. Furthermore, replacement of Trp2545, a conserved residue in the C‐terminal region throughout the PI‐kinase‐related kinase family, abolished the function of the mTOR kinase, both in vivo and in vitro. However, substitution of 32 C‐terminal residues of mTOR with those of ATM did not restore the mTOR function.ConclusionsThese findings define an indispensable role for the noncatalytic C‐terminal region of mTOR and indicate that, although this highly conserved region may be important throughout the PI‐kinase‐related kinase family, it is not functionally interchangeable within the family.
Two basic residues, Lys‐107 and Lys‐118, of RuvC resolvase are involved in critical contacts with the Holliday junction for its resolution
Authors: Yoshikawa Manabu , Iwasaki Hiroshi , Kinoshita Kengo , Shinagawa Hideo 
Published: March 2003

BackgroundCrystallographic and mutational studies of Escherichia coli RuvC Holliday junction resolvase have revealed that a catalytic site of each subunit is composed of four acidic residues at the bottom of the putative DNA‐binding cleft, whose surface contains eight basic residues.ResultsTo elucidate the functional roles of the basic residues on the cleft surface, we constructed a series of mutant ruvC genes and characterized their properties in vivo and in vitro. Among them, two RuvC mutants with a single alteration, K107A and K118A, were defective in UV‐repair and showed a dominant negative effect. The purified K107A and K118A proteins showed reduced binding activity to the junction DNA in the presence of Mg2+ under high salt conditions. Mn2+ increased both the junction binding and cleaving activities of the mutant proteins. In the absence of a divalent cation, the wild‐type, K107A and K118A proteins did not bind to junction DNA under high salt conditions, but the D7N mutant, with an alteration of the catalytic centre, was able to bind to the junction efficiently.ConclusionThe results presented here, in conjunction with previous crystallographic studies, suggest that the catalytic complex which is formed through interactions of acidic residues, Mg2+ and a cleavable phosphodiester bond, is stabilized by Lys‐107 and Lys‐118 via electrostatic interactions with the DNA backbone, a process which is critically important for the cleavage reaction to take place. One or two basic residues near the catalytic centre have also been found in other RNase H superfamily proteins, indicating that this is the conserved reaction mechanism in this superfamily.
Reduction of atherosclerosis despite hypercholesterolemia in lyn‐deficient mice fed a high‐fat diet
Authors: Miki Shunji , Horikawa Keisuke , Nishizumi Hirofumi , Suemura Masaki , Sato Bunzo , Yamamoto Masahiro , Takatsu Kiyoshi , Yamamoto Tadashi , Miki Yoshitsugu 
Published: March 2003

Oxidation and other modifications of serum low‐density lipoprotein (LDL) are associated with the development of atherosclerosis, and a scavenger receptor and CD40 signalling are also known to play important roles in the process. We previously showed that the Src family protein‐tyrosine kinase Lyn is physically and/or functionally associated with macrophage type‐I and type‐II class‐A scavenger receptors (MSR‐A) and CD40.In this study, we addressed whether Lyn is involved in the build‐up of serum lipid levels and in atherosclerotic changes. When fed a normal diet, lyn‐deficient mice had serum lipid levels that were no different from those of wild‐type mice. By contrast, lyn‐deficient mice fed a high‐fat diet showed serum lipid levels that were much higher than those seen in wild‐type mice. Curiously, however, the lyn‐deficient mice fed either diet showed no increase in incidence of atherosclerotic lesions compared with wild‐type mice. This may be partly explained by our data showing suppression of proliferation of peritoneal macrophages in response to oxidized LDL in the absence of Lyn, and failure of stimulation of the CD40 pathway in lyn‐deficient macrophages to induce expression of monocytic chemoattractant protein‐1 (MCP‐1), which is related to atherosclerosis.These results suggest that Lyn plays an important role in the metabolism of serum lipids and in the development of atherosclerotic lesions on high‐fat diets.
Monoallelic expresion of the odourant receptor gene and axonal projection of olfactory sensory neurones
Authors: Ishii Tomohiro , Serizawa Shou , Kohda Atsushi , Nakatani Hiroko , Shiroishi Toshihiko , Okumura Katsuzumi , Iwakura Yoichiro , Nagawa Fumikiyo , Tsuboi Akio , Sakano Hitoshi 
Published: March 2003

We have previously generated transgenic mice carrying the murine odourant receptor gene, MOR28, tagged with lacZ. In this animal, the endogenous MOR28 is differently tagged with GFP. It was found that the transgenic and endogenous MOR28 genes are expressed in a mutually exclusive manner and that the two sets of olfactory sensory neurones (OSNs), each expressing either the transgenic or the endogenous MOR28, project their axons to separate glomeruli.Our fluorescent in situ hybridization (FISH) revealed that the two endogenous alleles of MOR28 are also mutually excluded for their transcriptional activation. Therefore, we studied whether there would be any segregation in the projection of the two subsets of OSNs: one set expressing the paternal and the other expressing the maternal allele. It was found that the OSNs for both alleles shared the same glomerulus for their projection, but the projection targets were segregated within the glomerular structure.Two subsets of neurones expressing either the transgenic or the endogenous MOR28 target their axons to two separate glomeruli based on the differences in the genetic backgrounds, nature of tagging, and chromosomal locations. In contrast, neurones expressing a maternal or paternal allele share the same glomeruli, but tend to target to segregated areas within the glomerular structure. The segregation was more prominent with increased differences in the genetic background between the two alleles.
Cross talk of the interferon‐α/β signalling complex with gp130 for effective interleukin‐6 signalling
Authors: Mitani Yukiko , Takaoka Akinori , Kim Sun Hwa , Kato Yukiko , Yokochi Taeko , Tanaka Nobuyuki , Taniguchi Tadatsugu 
Published: March 2003

Background Signalling cross talk provides a molecular basis for modulating a given signalling pathway by another, and it is often critical for regulating cellular responses elicited by cytokines. Previously, we reported on the critical role of the IFN‐α/β signalling complex, generated by spontaneously produced IFN‐α/β, in efficient IFN‐γ signalling.Results In the present study, we have demonstrated that the IFN‐α/β signalling complex also contributes to efficient IL‐6 signalling. In fact, IL‐6‐induced activation of the Stat1 and Stat3 transcription factors is markedly diminished in the absence of the IFN‐α/β signalling complex. The induction of several target genes for these factors is also diminished, both in vitro and in vivo. We provide evidence that the cytoplasmic tyrosine residues of IFNAR‐1, which remains phosphorylated by a weak IFN‐α/β stimulation, provide docking sites for Stat1 and Stat3 to form homo‐ or heterodimers following IL‐6 stimulation. Furthermore, a chemical cross‐linking experiment revealed that IFNAR‐1 and gp130, a common signal transducer for the IL‐6 family of cytokines, exist in close proximity.Conclusions The constitutive weak IFN‐α/β signal provides a foundation for strong cellular responses to IL‐6, IFN‐γ, and possibly other cytokines. Our results also suggest the assembly of cytokine receptor subunits, which may represent a ‘receptosome’‐like structure, allowing the unique signalling cross talks to occur.
PKC1, a protein kinase C homologue of Saccharomyces cerevisiae, participates in microtubule function through the yeast EB1 homologue, BIM1
Authors: Hosotani Tomoki , Koyama Hirofumi , Uchino Masayuki , Miyakawa Tokichi , Tsuchiya Eiko 
Published: March 2003

Background RSC is a chromatin‐remodelling complex of Saccharomyces cerevisiae and essential for growth. Its catalytic subunit is encoded by the NPS1/STH1 gene. At the present time, little is known regarding the cellular function of RSC.Results To identify genes with functions related to NPS1, we screened high‐copy suppressor genes for the temperature‐ and thiabendazole (TBZ)‐sensitive mutant allele of NPS1, nps1‐105. Amongst the suppressors we cloned PKC1/STT1 and BIM1 that encoded a homologue of mammalian protein kinase C and a conserved microtubule binding protein homologous to human EB1, respectively. Both the temperature sensitive mutation of PKC1, stt1, and the bim1 null mutation caused synthetic growth defects with nps1‐105. A genetic analysis of the functional relationships between these genes revealed that PKC1 suppressed the defect of nps1‐105 through the BIM1 function but not by the activation of the MPK1/MAPK pathway. The stt1 mutation alone showed TBZ sensitivity and delayed the G2‐phase progression at semi‐permissive temperatures. Both of these stt1 phenotypes were suppressed by the over‐expression of BIM1. In addition, stt1 as well as nps1‐105, mis‐segregated a mini‐chromosome at frequencies higher than the wild‐type at a permissive temperature. The mis‐segregation was enhanced in the nps1‐105 stt1 double mutant.Conclusion These results suggest that Pkc1p plays a role which is relevant to microtubule functions and that this role is mediated by a hitherto unknown PKC signalling pathway and by Bim1p
Interaction of fission yeast ORC with essential adenine/thymine stretches in replication origins
Authors: Takahashi Tatsuro , Masukata Hisao 
Published: March 2003

Background Eukaryotic DNA replication is initiated from distinct regions on the chromosome. However, the mechanism for recognition of replication origins is not known for most eukaryotes. In fission yeast, replication origins are isolated as autonomously replicating sequences (ARSs). Multiple adenine/thymine clusters are essential for replication, but no short consensus sequences are found. In this paper, we examined the interaction of adenine/thymine clusters with the replication initiation factor ORC.Results The SpOrc1 or SpOrc2 immunoprecipitates (IPs) containing at least four subunits of SpORC, interacted with the ars2004 fragment, which is derived from a predominant replication origin on the chromosome. SpORC‐IPs preferentially interacted with two regions of the ars2004, which consist of consecutive adenines and AAAAT repeats and are essential for ARS activity. The nucleotide sequences required for the interaction with SpORC‐IPs correspond closely to those necessary for in vivo ARS activity.Conclusion Our results suggest that the SpORC interacts with adenine/thymine stretches, which have been shown to be the most important component in the fission yeast replication origin. The presence of multiple SpORC‐binding sites, with certain sequence variations, is characteristic for the fission yeast replication origins.
Math6, a bHLH gene expressed in the developing nervous system, regulates neuronal versus glial differentiation
Authors: Inoue Chihiro , Bae Soo‐Kyung , Takatsuka Kenji , Inoue Tomoyuki , Bessho Yasumasa , Kageyama Ryoichiro 
Published: March 2003

Background Whereas multiple basic helix‐loop‐helix (bHLH) genes are expressed in the developing nervous system, they account for the differentiation of only subsets of neurones, suggesting that there may be as‐yet unidentified bHLH genes.Results We have isolated a novel bHLH gene, designated Math6, a distant mammalian homologue of the Drosophila proneural gene atonal. Structural analysis of the Math6 gene demonstrated that the coding region is divided into three exons, whereas that of other atonal homologues is present in a single exon, indicating that the genomic structure of Math6 is unique among the atonal homologues. Math6 is initially expressed by neural precursor cells in the ventricular zone, but later by subsets of differentiating and mature neurones such as hippocampal neurones and cerebellar Purkinje cells. Mis‐expression of Math6 with retrovirus in the developing retina induced neurogenesis, while inhibiting gliogenesis, without affecting cell proliferation and death.Conclusions These results show that cells which would normally differentiate into glia adopted the neuronal fate by mis‐expression of Math6, indicating that Math6 promotes neuronal vs. glial fate determination in the nervous system.
Distinct regulatory mechanism for p70 S6 kinase β from that for p70 S6 kinase α
Authors: Minami Taichi , Hara Kenta , Oshiro Noriko , Ueoku Sachiko , Yoshino Ken‐ichi , Tokunaga Chiharu , Shirai Yasuhito , Saito Naoaki , Gout Ivan , Yonezawa Kazuyoshi 
Published: March 2003

Background A novel ribosomal S6 kinase, termed p70 S6 kinase β (p70β), has a highly homologous amino acid sequence to that of p70/p85 S6 kinase (p70α). This includes the critical phosphorylation sites, Thr252, Ser394 and Thr412 in p70α1, which correspond to Thr241, Ser383 and Thr401 in p70β1, respectively. However, the regulatory mechanism for p70β remains to be elucidated.Results We report here the expression and the mechanism of in vivo regulation of p70β. Two isoforms, p70β1 and p70β2, were expressed in a variety of tissues at a different level. p70β1 was mainly targeted to the nucleus, whereas p70β2 dispersed throughout the cytoplasm including nucleoplasm. The kinase activity of p70β1 was less sensitive to the inhibition induced by rapamycin, wortmannin and amino acid withdrawal than that of p70α. The portion of p70β activity inhibited by rapamycin was rescued by the rapamycin‐resistant mutant of the mammalian target of rapamycin (mTOR). Mutational analysis revealed that the phosphorylation of Thr241 and Thr401 in p70β1 was indispensable for the kinase activity. In contrast, a p70β1 mutant in which Ser383 was substituted with Gly (S383G) still retained nearly the half maximal activity. Sequential phosphorylation of wild‐type and S383G mutant of p70β1 with mTOR and 3‐phosphoinositide‐dependent protein kinase 1 (PDK1) in vitro synergistically activated their kinase activities.Conclusion These results indicate that p70β is regulated by the mTOR‐ and PDK1‐signalling pathways through a synergistic interaction between phosphorylated Thr241 and Thr401, while Ser383 plays minor role in their activation mechanism. Activated p70β may be less sensitive to dephosphorylation mediated by putative phosphatases activated by rapamycin, amino acid withdrawal, and probably wortmannin.
SPAL, a Rap‐specific GTPase activating protein, is present in the NMDA receptor‐PSD‐95 complex in the hippocampus
Authors: C. Roy Badal , Kohu Kazuyoshi , Matsuura Ken , Yanai Hiroyuki , Akiyama Tetsu 
Published: March 2003

AbstractBackground: The PSD‐95 family of proteins possesses multiple protein binding domains, including three PDZ domains, an SH3 domain, a HOOK domain and a guanylate kinase‐like (GK) domain. The PSD‐95 proteins function as scaffolding proteins that link ion channels such as the N‐methyl‐d‐aspartate‐receptors (NMDA‐Rs) with cytoskeletal networks and signalling molecules, thereby controlling synaptic plasticity and learning.Results: We found that the PSD‐95 family proteins interact via their GK domains with SPA‐1‐like protein (SPAL), a GTPase‐activating protein (GAP) that is specific for Rap1. SPAL was contained within the NMDA‐R‐PSD‐95 complex, and co‐localized with PSD‐95 and NMDA‐R at the synapses in cultured hippocampal neurones. Furthermore, NMDA stimulation induced the dephosphorylation of SPAL in cultured hippocampal neurones.Conclusion: Our findings suggest that SPAL may be involved in the NMDA‐mediated organization of cytoskeletal networks and signal transduction.
ADP stabilizes the human Rad51‐single stranded DNA complex and promotes its DNA annealing activity
Authors: Kim Hye‐Kyung , Morimatsu Katsumi , Nordén Bengt , Ardhammar Malin , Takahashi Masayuki 
Published: March 2003

AbstractBackground: Human Rad51 protein (HsRad51) is a homologue of Escherichia coli RecA protein, and involved in homologous recombination. These eukaryotic and bacterial proteins catalyse strand exchange between two homologous DNA molecules, each forming a complex with single‐stranded DNA (ssDNA) and ATP as the initial step. Both proteins hydrolyse ATP; however, the role of ATP hydrolysis appears to vary between the two proteins.Results: Measurements using the fluorescence ssDNA analogue, poly(1,N6‐etheno‐deoxyadenosine), indicate that ATP affects the HsRad51–ssDNA complex, promoting two conformational states: one transient, rather rigid transition state and a final more flexible state. While ADP lowers the affinity of RecA protein to ssDNA, it is found to rather stabilize the HsRad51–ssDNA complex. ADP does not activate the strand exchange by HsRad51 but instead stimulates annealing between complementary ssDNAs.Conclusions: The hydrolysis of ATP promotes a transition of the HsRad51–ssDNA complex from a stiff state to less stiff state. The first state may be important for the strand separation of dsDNA in the initial step of strand exchange, while the second state may be important for annealing in the next step. However, hydrolysis does not dissociate HsRad51 from DNA as a component step of its recycling.
Interaction between apterous and early expression of vestigial in formation of the dorso‐ventral compartments in the Drosophila wing disc
Authors: Delanoue Rénald , Zider Alain , Cossard Raynald , Dutriaux Annie , Silber Jöel 
Published: March 2003

AbstractBackground: Compartment formation is a developmental process that requires the existence of barriers against intermixing between cell groups. In the Drosophila wing disc, the dorso‐ventral (D/V) compartment boundary is defined by the expression of the apterous (ap) selector gene in the dorsal compartment. AP activity is under control of dLMO which destabilizes the formation of the AP–CHIP complex.Results: We report that D/V boundary formation in the wing disc also depends on early expression of vestigial (vg). Our data suggest that vg is already required for wing cell proliferation before D/V compartmentalization. In addition, we show that over‐expression of vg can, to some extent, rescue the effect of the absence of ap on D/V boundary formation. Early VG product regulates AP activity by inducing dLMO and thus indirectly regulating ap target genes such as fringe and the PSα1 and PSα2 integrins.Conclusion: Normal cell proliferation is necessary for ap expression at the level of the D/V boundary. This would be mediated by vg, which interacts in a dose‐dependent way with ap.
Sensitivity of reflection seismic data to oil‐column height in high‐porosity sandstones
Authors: Ryan‐Grigor Sarah , Sayers Colin M. 
Published: March 2003

Tuning is the effect of interference between the reflections from the top and bottom of a thin layer on the amplitude of the composite reflection. For a homogeneous sandstone reservoir containing an oil column overlying brine, interference between the reflection from the top reservoir and the oil/water contact is a function of the height of the oil column. If the properties of the sandstone do not vary across the oil/water contact, the SS, PS and SP reflection coefficients from the oil/water contact are small in comparison to the PP reflection coefficient. This allows analytic expressions for the effective PP and PS reflection coefficients from the reservoir to be derived that include all P‐wave multiples within the oil column. For a given source/receiver offset, the component of the wavevector inside the oil column normal to the interface is larger for the PPPP reflection than for the PPPS reflection, due to the asymmetry in the raypath for the PPPS reflection. The PPPS reflection is therefore useful for determining oil‐column heights larger than that discriminated by the PPPP reflection, especially when used at wider offsets.A convenient classification of the AVO response of hydrocarbon‐bearing sandstone reservoirs overlain by shale is the scheme of Rutherford and Williams. Class 1 sands have higher acoustic impedance for normal incidence than the overlying shale, Class 2 sands have nearly the same acoustic impedance as the shale and Class 3 sands have lower acoustic impedance. Synthetic shot gathers calculated for these three classes as a function of oil‐column height show that a combination of the PPPP and the PPPS amplitudes can be plotted as a tuning trajectory, which can be used to determine the oil‐column height. This method is most sensitive for reservoirs that belong to AVO classes 1 and 2, and therefore may be useful in AVO analysis of Class 1 and 2 reservoirs where the traditional AVO indicators (developed for Class 3 reservoirs) do not work very well. These results demonstrate the usefulness of shear waves recorded in the marine environment at wide offsets.