bluuebunny/crossref_metadata_2025_split huggingface dataset:
Pathways for repair of topoisomerase I covalent complexes in Saccharomyces cerevisiae
Authors: Pouliot Jeffrey J. , Robertson Carol A. , Nash Howard A. 
Published: March 2003

Background The covalent linkage between DNA and the active site tyrosine of topoisomerase I can be stabilized by chemotherapeutic agents, adjacent DNA lesions, or mutational defects in the topoisomerase itself. Following collision with a replication fork, the covalent complex can be converted to a double‐strand break. Tdp1, an enzyme that can hydrolyse the bond between topoisomerase I and DNA, is thought to be involved in the repair of these lesions, but little is known about how such repair is accomplished.Results Reaction kinetics with model substrates reveal that the catalytic efficiency of Saccharomyces cerevisiae Tdp1 is relatively poor when the scissile bond is located in the middle of a duplex, but much better when it is located at the end of a structure. Survival of yeast after induction of a toxic topoisomerase is substantially reduced by inactivation of the TDP1 gene. Comparison of survival of single and double mutants places TDP1 and RAD52 in the same epistasis group but TDP1 and RAD9 in different epistasis groups. In the absence of RAD9, inactivation of TDP1 has a significant effect on the survival of cells following exposure to camptothecin but is without consequence for the survival of agents that do not target topoisomerase I.Conclusions Tdp1 acts as a specific repair enzyme for topoisomerase I lesions. Rather than working at their earliest occurrence, the enzyme acts after covalent complexes have been converted to DSBs. A second repair pathway also exists that functions independently of Tdp1 but requires RAD9 function to efficiently repair topoisomerase I‐linked DSBs. The efficiency of these pathways differs for complexes induced with the chemotherapeutic agent camptothecin vs. those accumulated by mutant forms of topoisomerase I.
A deubiquitinating enzyme, UCH/CeUBP130, has an essential role in the formation of a functional microtubule‐organizing centre (MTOC) during early cleavage in C. elegans
Authors: Lee Jungsoo , Jee Changhoon , Lee Jin Il , Lee Myon Hee , Lee Moon Hee , Koo Hyeon‐Sook , Chung Chin Ha , Ahnn Joohong 
Published: March 2003

Background Deubiquitinating enzymes generate monomeric ubiquitin in protein degradation pathways and are known to be important for the early development in many organisms.Results RNA interference experiments targeted for a UBP homologue, UCH/CeUBP130, in C. elegans resulted in cell division defective embryos. Immunostaining localized UCH/CeUBP130 in the sperm and at the microtubule‐organizing centre (MTOC) during early cleavage. Furthermore, the embryonic lethal phenotype was rescued by mating with wild‐type males.Conclusions Since it is known that the MTOC in the fertilized embryo is contributed by sperm asters in C. elegans, we suggest that UCH/CeUBP130 and ubiquitin protein degradation pathways may be involved in microtubule‐based sperm aster formation. Therefore UCH/CeUBP130 is necessary for the formation of a functional MTOC in the fertilized embryo of C. elegans.
Translation ability of mitochondrial tRNAsSer with unusual secondary structures in an in vitro translation system of bovine mitochondria
Authors: Hanada Takao , Suzuki Tsutomu , Yokogawa Takashi , Takemoto‐Hori Chie , Sprinzl Mathias , Watanabe Kimitsuna 
Published: March 2003

Background Metazoan mitochondrial (mt) tRNAs are structurally quite different from the canonical cloverleaf secondary structure. The mammalian mt tRNASerGCU for AGY codons (Y = C or U) lacks the entire D arm, whereas tRNASerUGA for UCN codons (N = A, G, C or U) has an extended anti‐codon stem. It has been a long‐standing problem to prove experimentally how these tRNAsSer work in the mt translation system.Results To solve the above‐mentioned problem, we examined their translational abilities in an in vitro bovine mitochondrial translation system using transcripts of altered tRNASer analogues derived from bovine mitochondria. Both tRNASer analogues had almost the same ability to form ternary complexes with mt EF‐Tu and GTP. The D‐arm‐lacking tRNASer GCU analogue had considerably lower translational activity than the tRNASerUGA analogue and produced mostly short oligopeptides, up to a tetramer. In addition, tRNASerGCU analogue was disfavoured by the ribosome when other tRNAs capable of decoding the cognate codon were available.Conclusion Both mt tRNASerGCU and tRNASerUGA analogues with unusual secondary structure were found to be capable of translation on the ribosome. However, the tRNASerGCU analogue has some molecular disadvantage on the ribosome, which probably derives from the lack of a D arm.
A GyrB‐GyrA fusion protein expressed in yeast cells is able to remove DNA supercoils but cannot substitute eukaryotic topoisomerase II
Authors: Trigueros Sonia , Roca Joaquim 
Published: March 2003

AbstractBackground: Type II topoisomerases are a highly conserved class of enzymes which transport one double‐stranded DNA segment through a transient break in another. Whereas the eukaryotic enzymes are homodimers of a single polypeptide, their bacterial homologues are homodimers of two independently coded protein subunits. Unlike eukaryotic topoisomerase II and bacterial topoisomerase IV, DNA gyrase is a bacterial type II topoisomerase which specializes in intramolecular DNA transport.Results: We have fused the Escherichia coli coding sequences for the proteins GyrB and GyrA, which comprise DNA gyrase. This fusion was expressed in yeast cells and yielded the expected full‐length protein product. When it was expressed in Δtop1‐ top2‐4 yeast cells, the fusion protein compensated their slow growth and reverted their elevated chromosomal excision of ribosomal genes. Furthermore, it removed DNA positive supercoils. The fusion protein, however, was unable to complement the temperature‐dependent lethality of top2‐4 cells.Conclusion:Fusion of the E. coli GyrB and GyrA proteins leads to a catalytically active topoisomerase which compensates several phenotypic traits attributed to unconstrained DNA supercoiling in topoisomerase‐deficient cells. However, since the fusion protein cannot substitute for topoisomerase II, it may be efficient in intramolecular but not intermolecular DNA passage, resembling the catalytic properties of DNA gyrase.
CLC‐3 deficiency leads to phenotypes similar to human neuronal ceroid lipofuscinosis
Authors: Yoshikawa Momono , Uchida Shinichi , Ezaki Junji , Rai Tatemitsu , Hayama Atsushi , Kobayashi Katsuki , Kida Yujiro , Noda Masaki , Koike Masato , Uchiyama Yasuo , Marumo Fumiaki , Kominami Eiki , Sasaki Sei 
Published: March 2003

AbstractBackground: CLC‐3 is a member of the CLC chloride channel family and is widely expressed in mammalian tissues. To determine the physiological role of CLC‐3, we generated CLC‐3‐deficient mice (Clcn3–/–) by targeted gene disruption.Results: Together with developmental retardation and higher mortality, the Clcn3–/– mice showed neurological manifestations such as blindness, motor coordination deficit, and spontaneous hyperlocomotion. In histological analysis, the Clcn3–/– mice showed a pattern of progressive degeneration of the retina, hippocampus and ileal mucosa, which resembled the phenotype observed in cathepsin D knockout mice. The defect of cathepsin D results in a lysosomal accumulation of ceroid lipofuscin containing the mitochondrial F1F0 ATPase subunit c. In immunohistochemistry and Western blot analysis, we found that the subunit c was heavily accumulated in the lysosome of Clcn3–/– mice. Furthermore, we detected an elevation in the endosomal pH of the Clcn3–/– mice.Conclusions: These results indicated that the neurodegeneration observed in the Clcn3–/– mice was caused by an abnormality in the machinery which degrades the cellular protein and was associated with the phenotype of neuronal ceroid lipofuscinosis (NCL). The elevated endosomal pH could be an important factor in the pathogenesis of NCL.
Sexually dimorphic expression of Dax‐1 in the adrenal cortex
Authors: Mukai Tokuo , Kusaka Masatomo , Kawabe Ken , Goto Kiminobu , Nawata Hajime , Fujieda Kenji , Morohashi Ken‐ichirou 
Published: March 2003

AbstractBackground: The DAX‐1 (also known as AHC) gene encodes an unusual member of the nuclear receptor superfamily, and the gene product plays a pivotal role during gonadal and adrenal differentiation. Mutations of the human DAX‐1 gene cause X‐linked adrenal hypoplasia congenita associated with hypogonadotropic hypogonadism. However, the molecular mechanisms underlying the transcriptional regulation of the gene are not well understood.Results: Sexually dimorphic expression of Dax‐1 (NR0B1) in the adrenal cortex was observed by RT‐PCR, Western blotting and immunohistochemistry. The differential expression was abolished by gonadectomy and was restored again by sex steroid replacement. Our results suggested that the Dax‐1 gene transcription is suppressed by androgens and androgen receptor (AR/NR3C4). Dax‐1 gene transcription is regulated by Ad4BP/SF‐1 (NR5A1), therefore we investigated the functional correlation between A4BP/SF‐1 and AR. Interestingly, AR down‐regulated the Dax‐1 gene transcription mediated by Ad4BP/SF‐1 in the presence of the ligand. DNA binding by AR was not essential for the suppressive action, however the suppression seemed to be dependent on the promoter contexts. An interaction between Ad4BP/SF‐1 and AR was detected in the presence of the ligand for AR.Conclusion: The present study revealed that the expression of Dax‐1 in the adrenal cortex is regulated by androgen and the receptor. Interestingly, AR acts as a suppressor in the presence of the ligand through interaction with Ad4BP/SF‐1.
Distinct roles of SOX2, Pax6 and Maf transcription factors in the regulation of lens‐specific δ1‐crystallin enhancer
Authors: Muta Mayumi , Kamachi Yusuke , Yoshimoto Aki , Higashi Yujiro , Kondoh Hisato 
Published: March 2003

AbstractBackground: The eye lens provides a good model for the study of regulation of cell differentiation, in which lens‐specific δ1‐crystallin expression serves as an indicator of the differentiated state of the cells. It has been indicated that the SOX2, Pax6 and Maf proteins are the major regulators of lens cell differentiation. To clarify the individual roles of these transcription factors, we analysed their participation in regulation of the δ1‐crystallin enhancer.Results: We defined the major binding sites of SOX2, Pax6 and Maf transcription factors in the δ1‐crystallin enhancer and assessed the effect of mutations at these sites in the cultured lens epithelial cells and in developing lenses of transgenic mouse embryos. SOX2 (or SOX1/SOX3) is essential for activation of the enhancer under all conditions. Pax6 bound at the δEF3 site is required for activation of the enhancer, while Pax6 at the Pax6U site appears to be involved in the Pax6‐dependent suppression of the enhancer. In contrast, Maf proteins are only required for high enhancer activity in lens fibre cells.Conclusion: The distinct roles of these transcription factors in the regulation of δ1‐crystallin enhancer would tend to indicate their individual functions in lens differentiation. The activity of SOX2 and the related SOX1/3 is essential at all stages of lens development as transcriptional activators. Pax6, although it is required in all steps, probably exerts complex regulatory effects, since it possesses both the potential to activate and repress. The major function of Maf proteins presumably resides in the activation of the genes in lens fibre cells.
Novel family of CCCH‐type zinc‐finger proteins, MOE‐1, ‐2 and ‐3, participates in C. elegans oocyte maturation
Authors: Shimada Masumi , Kawahara Hiroyuki , Doi Hirofumi 
Published: March 2003

AbstractBackground: Oocyte maturation is an important prerequisite for the production of progeny. Although several germ‐line mutations have been reported, the precise mechanism by which the last step of oocyte maturation is controlled remains unclear. In Caenorhabditis elegans, CCCH‐type zinc‐finger proteins have been shown to be involved in germ cell formation, although their involvement in oocyte maturation has not been fully investigated.Results: Using a multiple RNAi technique, we have identified three novel redundant CCCH‐type zinc‐finger genes, named by us moe‐1, ‐2 (oma‐1, ‐2) and moe‐3, as a group related by functions and nucleotide sequence. Although a single RNAi of each moe gene was not effective, double or triple RNAi induced defects in oocyte maturation. We found that each moe transcript was expressed from the distal to proximal region of the gonad, while their corresponding proteins are accumulated exclusively in proximal oocytes, with a close association to germ granules. Although MOE‐2 protein is rapidly removed from germ granules after fertilization, we found that MOE‐2 associates with the centrosome‐peripheral structure in dividing blastomeres.Conclusions: Our results suggest that moe gene products are unique multifunctional proteins in terms of their redundancy and characteristic behaviour during the course of oocyte maturation. These gene products participate in processes in the final step of the meiotic cell cycle control, a novel function for CCCH‐type zinc‐finger family proteins thus far discovered.
Suppression of NF‐κB‐dependent gene expression by a hexamethylene bisacetamide‐inducible protein HEXIM1 in human vascular smooth muscle cells
Authors: Ouchida Rika , Kusuhara Masatoshi , Shimizu Noriaki , Hisada Tetsuya , Makino Yuichi , Morimoto Chikao , Handa Hiroshi , Ohsuzu Fumitaka , Tanaka Hirotoshi 
Published: March 2003

AbstractBackground: Neointima formation is a characteristic feature of atherosclerosis and post‐angioplasty restenosis, in which various soluble factors and mechanical injury stimulate signalling pathways in vascular smooth muscle cells (VSMC), promoting their migration and proliferation, and the eventual formation of the neointima. The transcription factor NF‐κB has been shown to play a pivotal role in this process. Hexamethylene bisacetamide, an inhibitor of VSMC proliferation, induces the mRNA expression of HEXIM1 (hexamethylene bisacetamide‐inducible protein 1). However, the protein expression and function of HEXIM1 remain unknown.Results: In the present study, we demonstrated that HEXIM1 localizes in the cytoplasm and nucleus, and its nuclear expression is restricted to discrete speckled areas. Treatment of VSMC with hexamethylene bisacetamide up‐regulated HEXIM1 expression, not only in mRNA but also protein levels. Moreover, HEXIM1 is shown to suppress the transcriptional activity of NF‐κB via its C‐terminal leucine‐rich domain. A glutathione‐S‐transferase pull down assay indicated that HEXIM1 interacts with the p65 subunit of NF‐κB. In VSMC, treatment with hexamethylene bisacetamide resulted in a down‐modulation of the transcription of NF‐κB target genes.Conclusion: We may therefore conclude that HEXIM1 plays an inhibitory role in NF‐κB‐dependent gene expression in VSMC and is the candidate of a novel therapeutic target for inhibition of VSMC proliferation.
CDK‐ and Cdc45‐dependent priming of the MCM complex on chromatin during S‐phase in Xenopus egg extracts: possible activation of MCM helicase by association with Cdc45
Authors: Masuda Taro , Mimura Satoru , Takisawa Haruhiko 
Published: March 2003

AbstractBackground: MCM and Cdc45 are required for the initiation and elongation stages of eukaryotic DNA replication. Recent studies show that a purified Mcm4/6/7 complex has DNA helicase activity. However, the biochemical function of the MCM complex and Cdc45 bound to chromatin has not been elucidated.Results: We have examined the biochemical properties of MCM proteins bound to chromatin fractions using Xenopus egg extracts. Immunoprecipitation of MCM proteins extracted under denaturing conditions reveals that all six subunits of MCM and Cdc45 form a tight complex following the initiation of DNA replication, and that both CDK activity and Cdc45 are essential for the complex formation. Chromatin immunoprecipitation of MCM proteins and Cdc45 shows that a complex containing MCM and Cdc45 has a DNA helicase activity which is dependent on CDK activity and Cdc45 in the extracts. Furthermore, both the complex and the helicase activity are resistant to treatment with phosphatase and high salt.Conclusions: Following the initiation of DNA replication, a tight MCM–Cdc45 complex is formed on chromatin and its formation is closely correlated with the DNA helicase activity of chromatin immunoprecipitates containing MCM and Cdc45. We propose that the tight MCM–Cdc45 complex functions as a replicative DNA helicase in vivo.
PML‐nuclear bodies are involved in cellular serum response
Authors: Matsuzaki Kazuhito , Minami Takeshi , Tojo Masahide , Honda Yoshiomi , Saitoh Noriko , Nagahiro Shinji , Saya Hideyuki , Nakao Mitsuyoshi 
Published: March 2003

AbstractBackground: Serum stimulation leads to the activation of various signal transduction pathways in cells, and the resultant signals are integrated into the serum response factor (SRF)‐dependent transcription of immediate‐early genes such as c‐fos.Results: To further characterize this response, we investigated the mechanism which controls serum response transcription in cultured human cells. Frequency of PML (promyelocytic leukaemia)‐nuclear bodies (NBs) formation increases shortly after serum stimulation, probably facilitating the interaction of SRF and CBP acetyltransferase at the NBs. PML modulates SRF‐mediated c‐fos promoter activities upon addition of serum to cells or expression of constitutively active Rho family GTPases. We mapped the region in the SRF that interacts with PML to the C‐terminal transactivation domain. An SRF mutant deleted of the transactivation domain neither co‐localizes with CBP in NBs nor fulfills its transcriptional role. Under conditions of serum stimulation, the formation of NBs coincides with the immediate‐early expression of the endogenous c‐fos gene in fibroblasts and in all‐trans retinoic acid‐treated acute promyelocytic leukaemia NB4 cells.Conclusion: These data provide an insight into the involvement of NBs in modulating the transcription of serum‐induced immediate‐early genes.
Electrical resistivity tomography survey for delineating uncharted mine galleries in West Bengal, India*
Authors: Maillol J.M. , Seguin M.‐K. , Gupta O.P. , Akhauri H.M. , Sen N. 
Published: March 2003

The history of subsidence, fires, flooding and other kinds of environmental hazards related to shallow coal workings in India goes back to colonial times some 300 years ago. As coal production accelerated in modern times, so did the environmental and socio‐economic drawbacks related to exploitation. In the mid‐1980s, a hydropneumatic sand‐stowing method was developed to fill in abandoned galleries but their exact location had to be known. Unfortunately, most of these old workings are uncharted and consequently large tracts of land cannot be stabilized. A research programme making use of integrated surface, borehole and cross‐hole geophysical methods was undertaken over a five‐year span to try to solve this problem. Surface geophysical methods, being cheaper and faster than their cross‐ and downhole counterparts, were used to cover larger areas on an exploratory basis, while cross‐hole methods were employed to locate more accurately one or a network of galleries to be perforated by drillhole(s) and used as a conduit for sand stowing.We report the results of one of the cross‐hole geophysical methods: electrical resistivity tomography (ERT). A pole–dipole configuration is used and both cross‐hole and surface‐borehole methodologies are tested. Forward modelling and inversion of synthetic data making use of downhole and surface physical and geometrical parameters are presented first. This phase is followed by the inversion of real data. It is concluded that ERT is not applicable for the detection of dry voids, but is effective in a waterlogged environment which is estimated to represent 85–90% of the cases. In waterlogged galleries, ERT is applicable in both cross‐hole and surface‐downhole modes, the latter allowing a larger surface coverage at low cost. ERT is thus a reliable geophysical tool to image water‐filled voids and an adequate technique to address environmental and geotechnical problems.
Increasing the vertical resolution of conventional sub‐bottom profilers by parametric equalization
Authors: Cobo P. , Ranz C. , Cervera M. 
Published: March 2003

ABSTRACTVertical resolution, i.e. the ability to resolve two close reflectors, is a crucial aspect of pulses used in geo‐acoustic exploration of sea sub‐bottoms. This paper deals with the problem of exploring the shallowest unconsolidated layers of the seafloor with conventional piezo‐electric sonar pulses. Such transducers do not have a sufficiently broad transmission response to enable them to radiate short high‐resolution pulses. Therefore, some kind of equalization process must be applied to broaden the transmission response. Here, inverse filtering is used to calculate the transducer driving waveform so that the subsequent acoustic pulse has a zero‐phase cosine‐magnitude nature. Within a specified bandwidth, this pulse has minimum length, i.e. maximum resolution.The method has been applied to compress the acoustic pulses radiated by two piezo‐electric transducers. In conventional performance, these transducers radiate narrowband pulses which contain several cycles at the natural resonance frequency. Under equalized driving, both transducers emit broadband pulses, with resolving power greatly increased, at the cost of some amplitude loss. That is, the pulses radiated by both transducers have been shortened from 1 ms (low‐frequency transducer) and 0.274 ms (high‐frequency transducer) in conventional performance to 0.13 ms and 0.038 ms in equalized mode, with amplitude losses of 33% and 56%, respectively. The great improvement in the resolution of this technique is demonstrated by comparing the synthetic echograms that should be obtained when exploring a wedge model using zero‐phase cosine‐magnitude pulses with conventional ping pulses.
Traveltime computation with the linearized eikonal equation for anisotropic media
Authors: Alkhalifah Tariq 
Published: March 2003

A linearized eikonal equation is developed for transversely isotropic (TI) media with a vertical symmetry axis (VTI). It is linear with respect to perturbations in the horizontal velocity or the anisotropy parameter η. An iterative linearization of the eikonal equation is used as the basis for an algorithm of finite‐difference traveltime computations. A practical implementation of this iterative technique is to start with a background model that consists of an elliptically anisotropic, inhomogeneous medium, since traveltimes for this type of medium can be calculated efficiently using eikonal solvers, such as the fast marching method. This constrains the perturbation to changes in the anisotropy parameter η (the parameter most responsible for imaging improvements in anisotropic media). The iterative implementation includes repetitive calculation of η from traveltimes, which is then used to evaluate the perturbation needed for the next round of traveltime calculations using the linearized eikonal equation. Unlike isotropic media, interpolation is needed to estimate η in areas where the traveltime field is independent of η, such as areas where the wave propagates vertically.Typically, two to three iterations can give sufficient accuracy in traveltimes for imaging applications. The cost of each iteration is slightly less than the cost of a typical eikonal solver. However, this method will ultimately provide traveltime solutions for VTI media. The main limitation of the method is that some smoothness of the medium is required for the iterative implementation to work, especially since we evaluate derivatives of the traveltime field as part of the iterative approach. If a single perturbation is sufficient for the traveltime calculation, which may be the case for weak anisotropy, no smoothness of the medium is necessary. Numerical tests demonstrate the robustness and efficiency of this approach.