bluuebunny/crossref_metadata_2025_split huggingface dataset:
Sed4p functions as a positive regulator of Sar1p probably through inhibition of the GTPase activation by Sec23p
Authors: Saito‐Nakano Yumiko , Nakano Akihiko 
Published: March 2003

Sar1p belongs to a unique subfamily of small GTPases and is essential for formation of the transport vesicles from the endoplasmic reticulum (ER) that are destined to the Golgi apparatus. To understand how the GTPase cycle of Sar1p is regulated, we screened for multicopy suppressors of sar1 ts mutants and identified SED4.Although deletion of sed4 alone shows no growth defect, sar1 Δsed4 double mutant cells are inviable. In the Δsed4 background, the suppression activity of SAR1 towards sec12 and sec16 is lost. These observations suggest that SED4 is a very close partner of SAR1 and imply that Sed4p may act to increase the active Sar1p in the cell. Over‐expression of SEC12 does not remedy the lethality of sar1 Δsed4. The purified cytoplasmic domain of Sed4p does not show a guanine nucleotide exchange (GEF) activity toward Sar1p nor increase the GEF activity of Sec12p. On the contrary, over‐expression of SED4 aggravates the ts growth of sec23 cells. The cytoplasmic domain of Sed4p weakly inhibits the GTPase‐activating (GAP) activity of Sec23p toward Sar1p. In a microsome‐based COPII binding assay, the binding of the GDP‐form mutant Sar1p (D32G) is lower on the Δsed4 microsomes than on the wild‐type membranes.We propose a model that Sed4p counteracts the GAP action of Sec23p on to Sar1p.
Sez4 gene encoding an elongation subunit of DNA polymerase ζ is required for normal embryogenesis
Authors: Kajiwara Kagemasa , O‐Wang Jiyang , Sakurai Takayuki , Yamashita Shunji , Tanaka Masafumi , Sato Masahiro , Tagawa Masatoshi , Sugaya Eiichi , Nakamura Kenji , Nakao Kazuki , Katsuki Motoya , Kimura Minoru 
Published: March 2003

Background Sez4 identified as a seizure‐activated gene shows a similarity to the yeast REV3 that encodes a catalytic subunit of the nonessential DNA polymerase ζ which is involved in error‐prone translesion synthesis. Although yeast REV3 homologues in mouse and human have recently been identified and characterized, their precise roles remain elusive.ResultsHere we investigated the role of mouse pol ζ by targeted inactivation of the Sez4 gene. The homozygous Sez4 mutants died around embryonic day (E) 10.5. This lethal effect was the result of developmental defects and apoptotic cell death within the embryo proper at the gastrulation stage, and it was partially rescued at E12.5 by the expression of a Sez4‐transgene. In wild‐type embryos, Sez4 transcripts were up‐regulated within the embryo proper from E7.5, correlating well with the lethal stage of Sez4‐inactivation.ConclusionOur findings indicate that Sez4 is essential for epiblast lineage‐specific development and suggests a requirement of mammalian DNA polymerase ζ in the survival of certain subcellular populations which are indispensable to normal embryogenesis.
Identification and functional analysis of the gene for type I myosin in fission yeast
Authors: Toya Mika , Motegi Fumio , Nakano Kentaro , Mabuchi Issei , Yamamoto Masayuki 
Published: March 2003

BackgroundType I myosin is highly conserved among eukaryotes, and apparently plays important roles in a number of cellular processes. In the budding yeast, two myosin I species have been identified and their role in F‐actin assembly has been inferred.ResultsWe cloned the fission yeast myo1 gene, which apparently encoded a myosin I protein. Disruption of myo1 was not lethal, but it caused growth retardation at high and low temperatures, sensitivity to a high concentration of KCl, and aberrance in cell morphology associated with an abnormal distribution of F‐actin patches. An abnormal deposition of cell wall materials was also seen. Homothallic myo1Δ cells could mate, but heterothallic myo1Δ cells were poor in conjugation. Myo1p was necessary for the encapsulation of spores. The tail domain of Myo1p was pivotal for its function. Calmodulin could bind to Myo1p through the IQ domain at the neck.ConclusionsMyo1p appears to control the redistribution of F‐actin patches during the cell cycle. Loss of Myo1p function is likely to slow down the actin assembly/disassembly process, which results in a failure of the actin cycle to catch up with other events in both the mitotic and meiotic cell cycles, including extension of the conjugation tubes.
Restricted feeding entrains liver clock without participation of the suprachiasmatic nucleus
Authors: Hara Reiko , Wan Keiko , Wakamatsu Hisanori , Aida Reiko , Moriya Takahiro , Akiyama Masashi , Shibata Shigenobu 
Published: March 2003

BackgroundThere are two main stimuli that entrain the circadian rhythm, the light‐dark cycle (LD) and restricted feeding (RF). Light‐induced entrainment requires induction of the Per1 and Per2 genes in the suprachiasmatic nucleus (SCN), the locus of a main oscillator. In this experiment, we determined whether RF resets the expression of circadian clock genes in the mouse liver with or without participation of the SCN.ResultsMice were allowed access to food for 4 h during the daytime (7 h advance of feeding time) under LD or constant darkness (DD). The peaks of mPer1, mPer2, D‐site‐binding protein (Dbp) and cholesterol 7α‐hydroxylase (Cyp7A) mRNA in the liver were advanced 6–12 h after 6 days of RF, whereas those in SCN were unaffected. The advance of mPer expression in the liver by RF was still observed in SCN‐lesioned mice. A 7 h advance in the LD cycle advanced the peaks of clock gene expression in both the liver and SCN, whereas, a shift in the LD did not move the phase of the liver clock when the shift was carried out under a fixed RF schedule during the night‐time.ConclusionsThese results suggest that restricted feeding strongly entrained the expression of circadian clock genes in the liver without the participation of an SCN clock function.
Induction of CspA, an E. coli major cold‐shock protein, upon nutritional upshift at 37 °C
Authors: Yamanaka Kunitoshi , Inouye Masayori 
Published: March 2003

BackgroundThe synthesis of CspA, the major cold‐shock protein of Escherichia coli, is dramatically induced upon cold shock. It was recently reported that there is massive presence of CspA under nonstress conditions, and it is thus claimed that CspA as the cold‐shock protein is a misnomer.ResultsHere, we re‐examined and confirmed that CspA is induced upon culture dilution at 37 °C. However, its induction level is one‐sixth of the cold‐shock‐induced level, clearly indicating that the major stress that induces CspA is cold shock. It was further found that CspA induction can be achieved not only by culture dilution but also by the simple addition of nutrients, and that it was almost completely abolished in the presence of rifampicin or nalidixic acid. Nutritional upshift causes the induction of only CspA but not other cold‐shock‐inducible CspA homologues. The amount of cspA mRNA rapidly and transiently increased by culture dilution, but its stability was not significantly changed.ConclusionsThese results suggest that CspA is a nutritional‐upshift stress protein as well as a cold‐shock stress protein, and that CspA induction following nutritional upshift may be due to transcriptional activation.
A member of the YER057c/yjgf/Uk114 family links isoleucine biosynthesis and intact mitochondria maintenance in Saccharomyces cerevisiae
Authors: Kim Jong‐Myong , Yoshikawa Hiroshi , Shirahige Katsuhiko 
Published: March 2003

Background Two paralogs, YIL051c and YER057c, in the Saccharomyces cerevisiae genome are members of the YER057c/Yigf/Uk114 family, which is highly conserved among Eubacteria, Archaea and Eukarya. Although the molecular function of this protein family is not clear, previous studies suggest that it plays a role in the regulation of metabolic pathways and cell differentiation.Results Yil051cp is 70% identical in amino acid sequence to Yer057cp, and differs in that the former is longer by 16 amino acids containing, in part, the mitochondrial targeting signal at the N‐terminus of the protein. An HA‐tagged protein of Yil051cp is localized strictly in mitochondria, while that of Yer057cp is found in both cytoplasm and nucleus. Disruption of YIL051c (yil051cδ) resulted in severe growth retardation in glucose medium due to isoleucine auxotroph, and no growth in glycerol medium due to the loss of mitochondria. An extract prepared from yil051cδ cells showed no transaminase activity for isoleucine, while that for valine or leucine was intact. Haploid yil051cδ cells newly isolated from the YIL051c/yil051cδ hetero‐diploids gradually lost mitochondrial DNA within 24 h in the absence of, but not in the presence of, an isoleucine. Mutants either requiring leucine (leu2–112) or isoleucine‐valine (bat1Δ, bat2Δ) in a YIL051c background showed no changes in mitochondrial DNA maintenance in the absence of requirements.Conclusions Based on these results, we named Yil051c as Ibm1 (Isoleucine Biosynthesis and Mitochondria maintenance1) and concluded that: (i) Ibm1p determines the specificity of isoleucine biosynthesis, probably at the transamination step, (ii) Ibm1p is required for the maintenance of mitochondrial DNA when isoleucine is deficient, and (iii) Isoleucine compensates for the lack of Ibm1p. Taken together, Ibm1p may act as a sensor for isoleucine deficiency as well as a regulator determining the specificity for branched amino acid transaminase.
Expression of ribosome modulation factor (RMF) in Escherichia coli requires ppGpp
Authors: Izutsu Kaori , Wada Akira , Wada Chieko 
Published: March 2003

Background During the transition from the logarithmic to the stationary phase, 70S ribosomes are dimerized into the 100S form, which has no translational activity. Ribosome Modulation Factor (RMF) is induced during the stationary phase and binds to the 50S ribosomal subunit, which directs the dimerization of 70S ribosomes. Unlike many other genes induced in the stationary phase, rmf transcription is independent of the sigma S. To identify the factors that regulate the growth phase‐dependent induction of rmf, mutant strains deficient in global regulators were examined for lacZ expression directed by the rmf promoter.Results Among mutants of defective global regulators, only ppGpp deficiency (relA‐spoT double mutant) drastically reduced the level of rmf transcription to less than 10% of that seen in the wild‐type. Neither RMF nor 100S ribosomes were detected in this mutant in the stationary phase. rmf transcription correlated well with cellular ppGpp levels during amino acid starvation, IPTG induction of Ptrc‐relA455 and in other mutants with artificially increased ppGpp levels. Although the growth rate also correlated inversely with both ppGpp levels and rmf transcription, the observation that the growth rates of the ppGpp‐deficient and wild‐type strains varied equivalently when grown on different media indicates that the link between rmf transcription and ppGpp levels is not a function of the growth rate.Conclusions ppGpp appears to positively regulate rmf transcription, at least in vivo. Thus, RMF provides a novel negative translational control by facilitating the formation of inactive ribosome dimers (100S) under the stringent circumstances of the stationary phase.
Chicken pineal clock genes: implication of BMAL2 as a bidirectional regulator in circadian clock oscillation
Authors: Okano Toshiyuki , Yamamoto Kazuyuki , Okano Keiko , Hirota Tsuyoshi , Kasahara Takaoki , Sasaki Momoko , Takanaka Yoko , Fukada Yoshitaka 
Published: March 2003

Background In a transcription/translation‐based autoregulatory feedback loop of vertebrate circadian clock systems, a BMAL1‐CLOCK heterodimer is a positive regulator for the transcription of the negative element gene Per. The chicken pineal gland represents a photosensitive clock tissue, but the pineal clock genes constituting the oscillator loop have been less well characterized.Results We identified expression of the Per2, Bmal1, Bmal2 and Clock genes in the chicken pineal gland. Messenger RNA levels of these genes exhibited overt circadian rhythms in the pineal cells, both in vivo and in culture. In vitro functional analyses revealed the formation of cBMAL1‐cCLOCK and cBMAL2‐cCLOCK heteromers. Both of the cBMAL‐cCLOCK heteromers activated E‐box element‐dependent transcription, which was negatively regulated by cPER2 in luciferase assays. Co‐expression of cCLOCK, cBMAL1 and cBMAL2 co‐operatively activated E‐box element‐dependent transcription, and a greater level of expression of cBMAL2 inhibited the activation. In the cultured pineal cells, an over‐expression of either cBMAL1 or cBMAL2 disrupted the circadian rhythm of melatonin production.Conclusion The functional characterization of the chicken pineal clock molecules supports the key roles of BMAL1, BMAL2 and CLOCK which contribute to the E‐box‐dependent transcriptional regulation in the circadian clock system.
Regulation of Ras and Rho small G proteins by SHP‐2
Authors: Kodama Atsuko , Matozaki Takashi , Shinohara Masahiko , Fukuhara Atsunori , Tachibana Kouichi , Ichihashi Masamitsu , Nakanishi Hiroyuki , Takai Yoshimi 
Published: March 2003

Background Hepatocyte growth factor/scatter factor (HGF/SF) induces cell scattering through the tyrosine kinase‐type HGF/SF receptor, c‐Met. We have previously shown that SHP‐2, a protein tyrosine phosphatase, positively regulates the HGF/SF‐induced cell scattering through modulating the activity of Rho to form stress fibres and focal adhesions. To further investigate the role of SHP‐2 in HGF/SF‐induced cell scattering, we have now examined the effect of a dominant active mutant of SHP‐2 (SHP‐2‐DA).Results Expression of SHP‐2‐DA markedly increased the formation of lamellipodia with ruffles, while it decreased the accumulation of E‐cadherin and β‐catenin at cell–cell adhesion sites in MDCK cells. In addition, expression of SHP‐2‐DA markedly enhanced cell scattering of MDCK cells in response to HGF/SF. Expression of SHP‐2‐DA induced the activation of MAP kinase without HGF/SF stimulation, whereas an inhibitor of MEK partly reversed the SHP‐2‐DA‐induced morphological phenotypes. Furthermore, expression of either a dominant‐active mutant of Rho or Vav2 also reversed the SHP‐2‐DA‐induced morphological phenotypes.Conclusion These results indicate that SHP‐2 plays a crucial role in the HGF/SF‐induced cell scattering through the regulation of two distinct small G proteins, Ras and Rho.
Tumour invasion and metastasis are promoted in mice deficient in tenascin‐X
Authors: Matsumoto Ken‐ichi , Takayama Noriyasu , Ohnishi Junji , Ohnishi Eriko , Shirayoshi Yasuaki , Nakatsuji Norio , Ariga Hiroyoshi 
Published: March 2003

Background Tenascin‐X (TNX) is a member of the tenascin family of large oligomeric glycoproteins of the extracellular matrix (ECM). To determine whether TNX plays a part in tumour invasion and metastasis and to disclose its normal physiological role, we disrupted its gene in mouse embryonic stem cells by homologous recombination and created mice deficient in TNX.Results TNX‐null mutant (TNX–/–) mice arose at normal frequency and showed no obvious defects during their adult life. However, when TNX–/– mice were subcutaneously inoculated in foot‐pads with a highly invasive and metastatic cell line, B16‐BL6 melanoma cells, the primary tumour size at 30 days after inoculation in the TNX–/– mice had increased by 1.2‐fold compared with that in wild‐type mice, and the invasion to the ankle and pulmonary metastasis in TNX–/– mice were also augmented by 2.2‐fold and 6.8‐fold, respectively, compared to those in wild‐type mice. To disclose the molecular mechanism(s) of the promotion of tumour invasion and metastasis in TNX–/– mice, we measured the protein levels of matrix metalloproteinases (MMPs), which are recognized as playing a key role in these events, in the foot‐pad homogenates of TNX–/– mice prior to the inoculation of melanoma cells. Gelatin zymography showed that the activities of proMMP‐2, active MMP‐2 and proMMP‐9 were significantly higher in TNX–/– mice than in wild‐type mice. Furthermore, a Northern blot analysis demonstrated that this increased activity of MMP‐2 in TNX–/– mice was due to the induced expression of MMP‐2 at the transcriptional level. The elevated expression of MMP‐2 and MMP‐9 resulted in decreased laminin levels, to less than half that of wild‐type mice in the homogenates of TNX–/– mice.Conclusions TNX deficiency led to an increase in the production of MMPs, and the increased activity of MMPs may result in the degradation of laminin. Consequently, the melanoma cells inoculated in TNX–/– mice might facilitate invasion and metastasis. These results imply that TNX is required for impeding the invasion and metastasis of tumour cells.
Sptrx‐2, a fusion protein composed of one thioredoxin and three tandemly repeated NDP‐kinase domains is expressed in human testis germ cells
Authors: Sadek Christine M. , Damdimopoulos Anastasios E. , Pelto‐Huikko Markku , Gustafsson Jan‐Åke , Spyrou Giannis , Miranda‐Vizuete Antonio 
Published: March 2003

Background Thioredoxins (Trx) are small redox proteins that function as general protein disulphide reductases and regulate several cellular processes such as transcription factor DNA binding activity, apoptosis and DNA synthesis. In mammalian organisms, thioredoxins are generally ubiquitously expressed in all tissues, with the exception of Sptrx‐1 which is specifically expressed in sperm cells.Results We report here the identification and characterization of a novel member of the thioredoxin family, the second with a tissue‐specific distribution in human sperm, termed Sptrx‐2. The Sptrx‐2 ORF (open reading frame) encodes for a protein of 588 amino acids with two different domains: an N‐terminal thioredoxin domain encompassing the first 105 residues and a C‐terminal domain composed of three repeats of a NDP kinase domain. The Sptrx‐2 gene spans about 51 kb organized in 17 exons and maps at locus 7p13‐14. Sptrx‐2 mRNA is exclusively expressed in human testis, mainly in primary spermatocytes, while Sptrx‐2 protein expression is detected from the pachytene spermatocytes stage onwards, peaking at round spermatids stage. Recombinant full‐length Sptrx‐2 expressed in bacteria displayed neither thioredoxin nor NDP kinase enzymatic activity.Conclusions The sperm specific expression of Sptrx‐2, together with its chromosomal assignment to a position reported as a potential locus for flagellar anomalies and male infertility phenotypes such as primary ciliary dyskinesia, suggests that it might be a novel component of the human sperm axonemal organization.
Requirement of the IFN‐α/β‐induced CXCR3 chemokine signalling for CD8+ T cell activation
Authors: Ogasawara Kouetsu , Hida Shigeaki , Weng Youmin , Saiura Akio , Sato Kojiro , Takayanagi Hiroshi , Sakaguchi Shinya , Yokochi Taeko , Kodama Tatsuhiko , Naitoh Makoto , De Martino Julie A. , Taniguchi Tadatsugu 
Published: March 2003

AbstractBackground: Activation of both CD4+ T and CD8+ T cells is triggered by the engagement of the T cell antigen receptor (TCR) with MHC/peptide complexes on antigen‐presenting cells. This process also requires other molecular interactions, which transmit co‐stimulatory signals to these T cells. To ensure an effective immune response, distinct T cell subsets may additionally employ unique mechanism(s) for efficient activation.Results: We here show that mutant CD8+ T cells lacking the IFN‐α/β signalling components are hyporesponsive to antigen stimulation in vitro. We further show that IFN‐α/β‐mediated signals are required for induction of the chemokines IP‐10/I‐TAC and their common receptor, CXCR3, and in turn provide evidence that CXCR3‐mediated signals indeed function in the activation and proliferation of CD8+ T cells, particularly for the CD44low naive phenotype cells.Conclusion: The CXCR3 chemokine system is regulated by IFN‐α/β in CD8+ T cells, and it is critical for the efficient cell activation. The present study therefore reveals a novel role of the IFN‐α/β–CXCR3 signalling cascade in CD8+ T cell activation.
Identification and characterization of tRNA (Gm18) methyltransferase from Thermus thermophilus HB8: domain structure and conserved amino acid sequence motifs
Authors: Hori Hiroyuki , Suzuki Tsutomu , Sugawara Kazumasa , Inoue Yorinao , Shibata Takehiko , Kuramitsu Seiki , Yokoyama Shigeyuki , Oshima Tairo , Watanabe Kimitsuna 
Published: March 2003

AbstractBackground: Transfer RNAs from an extreme thermophile, Thermus thermophilus, commonly possess 2′‐O‐methylguanosine at position 18 (Gm18) in the D‐loop. This modification is post‐transcriptionally introduced by tRNA (Gm18) methyltransferase.Results: Partial amino acid sequence data were obtained from purified T. thermophilus tRNA (Gm18) methyltransferase by peptide sequencing and mass spectrometry. The sequence data were used to screen the T. thermophilus genome database currently in progress, resulting in the identification of the corresponding gene. Purified recombinant enzyme showed a strict specificity for methylation at the 2′‐OH of G18 in tRNA. Sequence alignment with other known or putative methyltransferases elucidates that tRNA (Gm18) methyltransferases have specific conserved region as well as three consensus motifs found in RNA ribose 2′‐O‐methyltransferases. The enzyme truncated at its N and C termini by limited tryptic digestion still retained binding activity for S‐adenosyl‐l‐homocysteine, but lost the catalytic activity.Conclusion: This is the first report describing the identification of a methyltransferase gene of the trmH family through the analysis of a purified protein. Further, our results indicate that a restricted region(s) in the terminal amino acid residues of T. thermophilus tRNA (Gm18) methyltransferase are responsible for tRNA recognition and a main part of the enzyme is allocated for a catalytic core.
Identification of a BMP‐responsive element in Id1, the gene for inhibition of myogenesis
Authors: Katagiri Takenobu , Imada Mana , Yanai Takeshi , Suda Tatsuo , Takahashi Naoyuki , Kamijo Ryutaro 
Published: March 2003

AbstractBackground: Bone morphogenetic protein‐2 (BMP‐2) stimulates osteoblast differentiation, but inhibits myogenic differentiation in C2C12 myoblasts. BMP‐2 induces transcription of Id1, an inhibitor for myogenesis, within 1 h in the cells. To examine the molecular mechanism of the action of BMP‐2, we analysed a BMP‐2‐responsive element (BRE) in the 5′ flanking region of the human Id1 gene.Results: A GC‐rich region between −985 bp and −957 bp of the human Id1 gene was identified as a BRE. The BRE containing promoter activity was stimulated by BMP‐2 or by constitutively active BMP receptors (BMPR‐IA and BMPR‐IB). The stimulation was blocked by co‐transfecting with dominant negative BMPR‐IA or Smad7. A unique DNA–protein complex was induced in response to BMP‐2 on the BRE. The complex induced by BMP‐2 contained Smad1 and Smad4, possibly as a complex of both Smads. BMP‐2 failed to stimulate the expression of Id1 mRNA in Smad4‐deficient cells. Over‐expression of Smad4, but not Smad1, stimulated the Id1 reporter activity and the expression of endogenous Id1 mRNA in Smad4‐deficient cells.Conclusion: Signalling of BMP‐2 to stimulate the expression of Id1 would be transduced by BMPR‐IA and mediated by Smad1 and Smad4, both of which form a complex on the 29 bp GC‐rich element.