bluuebunny/crossref_metadata_2025 huggingface dataset:
Adaptor protein Shc undergoes translocation and mediates up‐regulation of the tyrosine kinase c‐Src in EGF‐stimulated A431 cells
Authors: Sato Ken‐ichi , Kimoto Miwa , Kakumoto Miki , Horiuchi Dai , Iwasaki Tetsushi , Tokmakov Alexander A. , Fukami Yasuo
Published: March 2003

Background

Shc is the adaptor protein that exists in three isoforms, P46, P52 and P66, and acts as a bridge between activated cell surface receptors and downstream signalling molecules which act in extracellular signal‐regulated cell events such as cell cycle progression. In our previous studies, Shc was shown to be a substrate of the tyrosine kinase c‐Src in vitro and in vivo.

Results

Using green fluorescent protein‐fusion Shc (GFP‐Shc), we have shown that following epidermal growth factor (EGF) stimulation of A431 cells, all Shc isoforms were rapidly recruited from the cytoplasm to the plasma membrane (within 5 min) and then redistributed to the cytoplasmic vesicle structures (in the next 10–20 min). Indirect immunofluorescent study demonstrated that all Shc isoforms co‐localize with EGF receptor (EGFR) and activated c‐Src in both plasma membranes and cytoplasmic vesicle structures. Our previous study has shown that EGF induces the indirect association of EGFR and c‐Src and activation of c‐Src in A431 cells. An immunoprecipitation study demonstrated that the EGFR–Src association and c‐Src activation are augmented in cells expressing GFP‐Shc P52 or P66, but not P46. In addition, P52 and P66, but not P46, are in association with EGFR‐Src complex. We also found that EGFR and Shc can be dissociated from c‐Src by the addition of a synthetic peptide that corresponds to the autophosphorylation site of c‐Src. Interestingly, the peptide‐induced dissociation of the complex was not affected by the tyrosine phosphorylation state of the peptide.

Conclusion

These results demonstrated a dynamic subcellular movement of Shc in response to EGF, and suggested a hitherto unknown scheme whereby Shc can work not only as a substrate of c‐Src but also as a mediator of the EGF‐induced activation of c‐Src in an isoform‐specific manner.
The absence of ribonuclease H1 or H2 alters the sensitivity of Saccharomyces cerevisiae to hydroxyurea, caffeine and ethyl methanesulphonate: implications for roles of RNases H in DNA replication and repair
Authors: Arudchandran Arulvathani , Cerritelli Susana , Narimatsu Scott , Itaya Mitsuhiro , Shin Deug‐Yong , Shimada Yuji , Crouch Robert
Published: March 2003

Background

RNA of RNA‐DNA hybrids can be degraded by ribonucleases H present in all organisms including the eukaryote Saccharomyces cerevisiae. Determination of the number and roles of the RNases H in eukaryotes is quite feasible in S. cerevisiae.

Results

Two S. cerevisiae RNases H, related to Escherichia coli RNase HI and HII, are not required for growth under normal conditions, yet, compared with wild‐type cells, a double‐deletion strain has an increased sensitivity to hydroxyurea (HU) and is hypersensitive to caffeine and ethyl methanesulphonate (EMS). In the absence of RNase H1, RNase H2 activity increases, and cells are sensitive to EMS but not HU and are more tolerant of caffeine; the latter requires RNase H2 activity. Cells missing only RNase H2 exhibit increased sensitive to HU and EMS but not caffeine

Conclusions

Mutant phenotypes infer that some RNA‐DNA hybrids are recognized by both RNases H1 and H2, while other hybrids appear to be recognized only by RNase H2. Undegraded RNA‐DNA hybrids have an effect when DNA synthesis is impaired, DNA damage occurs or the cell cycle is perturbed by exposure to caffeine suggesting a role in DNA replication/repair that can be either beneficial or detrimental to cell viability.
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.

Results

Here 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.

Conclusion

Our 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.
Histone acetylases—versatile players
Authors: Nakatani Yoshihiro
Published: March 2003

Genomic DNA in eukaryotes is tightly packed in the form of a highly ordered chromatin structure. In view of this tight packing, one of the most important questions in biology is how the transcriptional machinery regulates target genes in chromatin. Reversible modification of histones by acetylation is involved in transcriptional activation as well as repression in chromatin contexts. Recent studies with highly purified histone acetylases have provided insights into the mechanisms whereby acetylases contribute to transcriptional control. Furthermore, they suggest the possibility that histone acetylases could play roles in various forms of DNA metabolism as well as in transcription in chromatin contexts.
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

Background

Type 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.

Results

We 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.

Conclusions

Myo1p 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

Background

There 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.

Results

Mice 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.

Conclusions

These 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

Background

The 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.

Results

Here, 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.

Conclusions

These 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

Background: 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

Background: 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.
The reaction‐diffusion system: a mechanism for autonomous pattern formation in the animal skin
Authors: Kondo Shigeru
Published: March 2003

How do animals acquire their various skin patterns? Although this question may seem easy, in fact it is very difficult to answer. The problem is that most animals have no related structures under the skin; therefore, the skin cells must form the patterns without the support of a prepattern. Recent progress in developmental biology has identified various molecular mechanisms that function in setting the positional information needed for the correct formation of body structure. None of these can explain how skin pattern is formed, however, because all such molecular mechanisms depend on the existing structure of the embryo. Although little is known about the underlying molecular mechanism, many theoretical studies suggest that the skin patterns of animals form through a reaction‐diffusion system—a putative ‘wave’ of chemical reactions that can generate periodic patterns in the field. This idea had remained unaccepted for a long time, but recent findings on the skin patterns of fish have proved that such waves do exist in the animal body. In this review, we explain briefly the principles of the reaction‐diffusion mechanism and summarize the recent progress made in this area.
Control of DNA replication licensing in a cell cycle
Authors: Nishitani Hideo , Lygerou Zoi
Published: March 2003

To maintain genome integrity in eukaryotes, DNA must be duplicated precisely once before cell division occurs. A process called replication licensing ensures that chromosomes are replicated only once per cell cycle. Its control has been uncovered by the discovery of the CDKs (cyclin dependent kinases) as master regulators of the cell cycle and the initiator proteins of DNA replication, such as the Origin Recognition Complex (ORC), Cdc6/18, Cdt1 and the MCM complex. At the end of mitosis, the MCM complex is loaded on to chromatin with the aid of ORC, Cdc6/18 and Cdt1, and chromatin becomes licensed for replication. CDKs, together with the Cdc7 kinase, trigger the initiation of replication, recruiting the DNA replicating enzymes on sites of replication. The activated MCM complex appears to play a key role in the DNA unwinding step, acting as a replicating helicase and moves along with the replication fork, at the same time bringing the origins to the unlicensed state. The cycling of CDK activity in the cell cycle separates the two states of replication origins, the licensed state in G1‐phase and the unlicensed state for the rest of the cell cycle. Only when CDK drops at the completion of mitosis, is the restriction on licensing relieved and a new round of replication is allowed. Such a CDK‐regulated licensing control is conserved from yeast to higher eukaryotes, and ensures that DNA replication takes place only once in a cycle. Xenopus laevis and mammalian cells have an additional system to control licensing. Geminin, whose degradation at the end of mitosis is essential for a new round of licensing, has been shown to bind Cdt1 and negatively regulate it, providing a new insight into the regulation of DNA replication in higher eukaryotes.
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

Background: 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.
Two major Smad pathways in TGF‐β superfamily signalling
Authors: Miyazawa Keiji , Shinozaki Masahiko , Hara Takane , Furuya Toshio , Miyazono Kohei
Published: March 2003

Members of the transforming growth factor‐β (TGF‐β) superfamily bind to two different serine/threonine kinase receptors, i.e. type I and type II receptors. Upon ligand binding, type I receptors specifically activate intracellular Smad proteins. R‐Smads are direct substrates of type I receptors; Smads 2 and 3 are specifically activated by activin/nodal and TGF‐β type I receptors, whereas Smads 1, 5 and 8 are activated by BMP type I receptors. Nearly 30 proteins have been identified as members of the TGF‐β superfamily in mammals, and can be classified based on whether they activate activin/TGF‐β‐specific R‐Smads (AR‐Smads) or BMP‐specific R‐Smads (BR‐Smads). R‐Smads form complexes with Co‐Smads and translocate into the nucleus, where they regulate the transcription of target genes. AR‐Smads bind to various proteins, including transcription factors and transcriptional co‐activators or co‐repressors, whereas BR‐Smads interact with other proteins less efficiently than AR‐Smads. Id proteins are induced by BR‐Smads, and play important roles in exhibiting some biological effects of BMPs. Understanding the mechanisms of TGF‐β superfamily signalling is thus important for the development of new ways to treat various clinical diseases in which TGF‐β superfamily signalling is involved.
ATPase/helicase motif mutants of Escherichia coli PriA protein essential for recombination‐dependent DNA replication
Authors: Tanaka Taku , Taniyama Chika , Arai Ken‐Ichi , Masai Hisao
Published: March 2003

Backgrounds: PriA protein, a DEXH‐type helicase with C2C2 zinc‐finger motifs, plays essential roles in RecA‐dependent modes of Escherichia coli chromosomal DNA replication, namely inducible and constitutive stable DNA replication (iSDR and cSDR respectively, which may be initiated from a D‐loop or R‐loop structure), and in repair of double‐stranded DNA breaks generated by various genotoxic agents or spontaneously during the course of DNA replication. However, the roles of ATPase/DNA helicase activities in functions of PriA are not well understood.

Results: We have generated and characterized mutants of PriA protein carrying amino acid substitutions in its conserved ATPase/DNA helicase motifs, namely the Walker A, B and QXXGRXGR motifs. All these mutants were deficient in ATP hydrolysis and DNA helicase activities, but showed wild‐type levels of D‐loop DNA binding, except for the Walker B mutant which showed reduced DNA binding activity, suggesting that the helicase motifs are not directly involved in the DNA binding activity of PriA protein. They also rescued the low viability and UV‐sensitivity of priA null cells. However, they did not rescue iSDR or cSDR—alternative modes of chromosomal DNA replication of the E. coli genome dependent on recombination functions—to the full extent.

Conclusions: ATPase/DNA helicase activities of PriA protein are required for full‐level DNA synthesis in recombination‐dependent modes of DNA replication in E. coli.
ATP‐induced hexameric ring structure of the cyanobacterial circadian clock protein KaiC
Authors: Hayashi Fumio , Suzuki Hirofumi , Iwase Ryo , Uzumaki Tatsuya , Miyake Asako , Shen Jian‐Ren , Imada Katsumi , Furukawa Yukio , Yonekura Koji , Namba Keiichi , Ishiura Masahiro
Published: March 2003

Background: KaiA, KaiB and KaiC are cyanobacterial circadian clock proteins. KaiC contains two ATP/GTP‐binding Walker's motif As, and mutations in these regions affect the clock oscillations.

Results: ATP induced the hexamerization of KaiC. The Km value for the ATP for the hexamerization was 1.9 µm. Triphosphate nucleotides bound to the two Walker's motif As, and their binding functioned cooperatively for the hexamerization. An unhydrolysable substrate, 5′‐adenylylimidodiphosphate (AMPPNP), also induced the hexamerization, indicating that nucleotide binding, but not its hydrolysis, is essential for the hexamerization. Mutations in each of the two Walker's motif As that affect the clock phenotype increased the Km value for ATP and inhibited the hexamerization. Thus, the KaiC hexamerization seems to be necessary for its clock function. The KaiC hexamer has the shape of a hexagonal pot with a diameter and height of approximately 100 Å and with a relatively large cavity (73 Å deep and 18–34 Å wide) inside. This pot‐shaped structure suggests that KaiC functions in a similar manner to F1‐ATPase, helicase or ATP‐dependent protease/chaperon, all of which have dynamic activities inside the central cavity of their hexameric rings.

Conclusion: ATP‐induced KaiC hexamerization is necessary for the clock function of KaiC.
Background velocity estimation using non‐linear optimization for reflection tomography and migration misfit
Authors: Varela Carlos L. , Stoffa Paul L. , Sen Mrinal K.
Published: March 2003

We show that it is possible to estimate the background velocity for prestack depth migration in 2D laterally varying media using a non‐linear optimization technique called very fast simulated annealing (VFSA). We use cubic splines in the velocity model parametrization and make use of either successive pairs of shot gathers or several constant‐offset sections as input data for the inversion. A Kirchhoff summation scheme based on first‐arrival traveltimes is used to migrate/model the input data during the velocity analysis. We evaluate and compare two different measures of error. The first is defined in the recorded data or (x,t) domain and is based on a reflection‐tomography criterion. The second is defined in the migrated data or (x,z) domain and is based on a migration‐misfit criterion. Depth relaxation is used to improve the convergence and quality of the velocity analysis while simultaneously reducing the computational cost. Further, we show that by coarse sampling in the offset domain the method is still robust.

Our non‐linear optimization approach to migration velocity analysis is evaluated for both synthetic and real seismic data. For the velocity‐analysis method based on the reflection‐tomography criterion, traveltimes do not have to be picked. Similarly, the migration‐misfit criterion does not require that depth images be manually compared. Interpreter intervention is required only to restrict the search space used in the velocity‐analysis problem. Extension of the proposed schemes to 3D models is straightforward but practical only for the fastest available computers.
Relationship between P‐ and S‐wave velocities and geological properties of near‐surface sediments of the continental slope of the Barents Sea
Authors: Ayres Arthur , Theilen Friedrich
Published: March 2003

Seismic velocities (Vp and Vs) of compressional (P‐) and shear (S‐) waves are important parameters for the characterization of marine sediments with respect to their sedimentological and geotechnical properties. P‐ and S‐wave velocity data of near‐surface marine sediments (upper 9 m) of the continental slope of the Barents Sea are analysed and correlated to sedimentological and geotechnical properties. The results show that the S‐wave velocity is much more sensitive to changes in lithology and mechanical properties than the P‐wave velocity, which is characterized by a narrow range of values. The correlation coefficients between S‐wave velocity and silt and clay content, wet bulk density, porosity, water content and shear strength are higher than 0.5 while the correlation coefficients of P‐wave velocity and the same parameters are always lower than 0.4. Although the relationship between Vs and clay content has been widely described, the data show that Vs is better correlated with silt content than with clay content for the sediments of the area investigated. However, they show different trends. While Vs increases with increasing clay content, it decreases with increasing silt content.
Non‐equilibrium compaction and abnormal pore‐fluid pressures: effects on rock properties1
Authors: Carcione José M. , Gangi Anthony F.
Published: March 2003

Knowledge of pore pressure using seismic data will help in planning the drilling process to control potentially dangerous abnormal pressures. Various physical processes cause anomalous pressures on an underground fluid. Non‐equilibrium compaction is a significant process of overpressure generation. This occurs when the sedimentation rate is so rapid that the pore fluids do not have a chance to ‘escape’ from the pore space.

The model assumes a closed system and that the pore space is filled with water and hydrocarbon in a liquid state. Balancing mass and volume fractions yields the fluid pressure versus time of deposition and depth of burial. Thermal effects are taken into account. The pore pressure, together with the confining pressure, determines the effective pressure which, in turn, determines the bulk moduli of the rock matrix.

We assume a sandstone saturated with hydrocarbons and water, for which calibration of the model with experimental data is possible. The seismic velocities and attenuation factors are computed by using Biot’s theory of dynamic poroelasticity and the generalized linear solid. The example shows that the formation can be overpressured or underpressured depending on the properties of the saturating fluid. Wave velocities and quality factors decrease with decreasing differential pressure. The effect is important below approximately 20 MPa. The model is in good agreement with experimental data for Berea sandstone and provides a tool for predicting pore pressure from seismic attributes.
Constraints in 3D gravity inversion
Authors: Boulanger Olivier , Chouteau Michel
Published: March 2003

A three‐dimensional (3D) inversion program is developed to interpret gravity data using a selection of constraints. This selection includes minimum distance, flatness, smoothness and compactness constraints, which can be combined using a Lagrangian formulation. A multigrid technique is also implemented to resolve separately large and short gravity wavelengths. The subsurface in the survey area is divided into rectangular prismatic blocks and the problem is solved by calculating the model parameters, i.e. the densities of each block. Weights are given to each block depending on depth, a priori information on density and the density range allowed for the region under investigation. The present computer code is tested on modelled data for a dipping dike and multiple bodies. Results combining different constraints and a weight depending on depth are shown for the dipping dike. The advantages and behaviour of each method are compared in the 3D reconstruction. Recovery of geometry (depth, size) and density distribution of the original model is dependent on the set of constraints used. From experimentation, the best combination of constraints for multiple bodies seems to be flatness and a minimum volume for the multiple bodies. The inversion method is tested on real gravity data from the Rouyn‐Noranda (Quebec) mining camp. The 3D inversion model for the first 10 km is in agreement with the known major lithological contacts at the surface; it enables the determination of the geometry of plutons and intrusive rocks at depth.
2D inversion of refraction traveltime curves using homogeneous functions
Authors: Piip V.B.
Published: March 2003

A method using simple inversion of refraction traveltimes for the determination of 2D velocity and interface structure is presented. The method is applicable to data obtained from engineering seismics and from deep seismic investigations. The advantage of simple inversion, as opposed to ray‐tracing methods, is that it enables direct calculation of a 2D velocity distribution, including information about interfaces, thus eliminating the calculation of seismic rays at every step of the iteration process. The inversion method is based on a local approximation of the real velocity cross‐section by homogeneous functions of two coordinates. Homogeneous functions are very useful for the approximation of real geological media. Homogeneous velocity functions can include straight‐line seismic boundaries. The contour lines of homogeneous functions are arbitrary curves that are similar to one another. The traveltime curves recorded at the surface of media with homogeneous velocity functions are also similar to one another. This is true for both refraction and reflection traveltime curves. For two reverse traveltime curves, non‐linear transformations exist which continuously convert the direct traveltime curve to the reverse one and vice versa. This fact has enabled us to develop an automatic procedure for the identification of waves refracted at different seismic boundaries using reverse traveltime curves. Homogeneous functions of two coordinates can describe media where the velocity depends significantly on two coordinates. However, the rays and the traveltime fields corresponding to these velocity functions can be transformed to those for media where the velocity depends on one coordinate. The 2D inverse kinematic problem, i.e. the computation of an approximate homogeneous velocity function using the data from two reverse traveltime curves of the refracted first arrival, is thus resolved. Since the solution algorithm is stable, in the case of complex shooting geometry, the common‐velocity cross‐section can be constructed by applying a local approximation. This method enables the reconstruction of practically any arbitrary velocity function of two coordinates. The computer program, known as godograf, which is based on this theory, is a universal program for the interpretation of any system of refraction traveltime curves for any refraction method for both shallow and deep seismic studies of crust and mantle. Examples using synthetic data demonstrate the accuracy of the algorithm and its sensitivity to realistic noise levels. Inversions of the refraction traveltimes from the Salair ore deposit, the Moscow region and the Kamchatka volcano seismic profiles illustrate the methodology, practical considerations and capability of seismic imaging with the inversion method.
A new direction for shallow refraction seismology: integrating amplitudes and traveltimes with the refraction convolution section
Authors: Palmer Derecke
Published: March 2003

The refraction convolution section (RCS) is a new method for imaging shallow seismic refraction data. It is a simple and efficient approach to full‐trace processing which generates a time cross‐section similar to the familiar reflection cross‐section. The RCS advances the interpretation of shallow seismic refraction data through the inclusion of time structure and amplitudes within a single presentation. The RCS is generated by the convolution of forward and reverse shot records. The convolution operation effectively adds the first‐arrival traveltimes of each pair of forward and reverse traces and produces a measure of the depth to the refracting interface in units of time which is equivalent to the time‐depth function of the generalized reciprocal method (GRM). Convolution also multiplies the amplitudes of first‐arrival signals. To a good approximation, this operation compensates for the large effects of geometrical spreading, with the result that the convolved amplitude is essentially proportional to the square of the head coefficient. The signal‐to‐noise (S/N) ratios of the RCS show much less variation than those on the original shot records. The head coefficient is approximately proportional to the ratio of the specific acoustic impedances in the upper layer and in the refractor. The convolved amplitudes or the equivalent shot amplitude products can be useful in resolving ambiguities in the determination of wave speeds. The RCS can also include a separation between each pair of forward and reverse traces in order to accommodate the offset distance in a manner similar to the XY spacing of the GRM. The use of finite XY values improves the resolution of lateral variations in both amplitudes and time‐depths. The use of amplitudes with 3D data effectively improves the spatial resolution of wave speeds by almost an order of magnitude. Amplitudes provide a measure of refractor wave speeds at each detector, whereas the analysis of traveltimes provides a measure over several detectors, commonly a minimum of six. The ratio of amplitudes obtained with different shot azimuths provides a detailed qualitative measure of azimuthal anisotropy and, in turn, of rock fabric. The RCS facilitates the stacking of refraction data in a manner similar to the common‐midpoint methods of reflection seismology. It can significantly improve S/N ratios.Most of the data processing with the RCS, as with the GRM, is carried out in the time domain, rather than in the depth domain. This is a significant advantage because the realities of undetected layers, incomplete sampling of the detected layers and inappropriate sampling in the horizontal rather than the vertical direction result in traveltime data that are neither a complete, an accurate nor a representative portrayal of the wave‐speed stratification. The RCS facilitates the advancement of shallow refraction seismology through the application of current seismic reflection acquisition, processing and interpretation technology.