bluuebunny/crossref_metadata_2025 huggingface dataset:
Design of Two-Dimensional Ultrasonic Phased Array Transducers
Authors: Mondal Shyamal C. , Wilcox Paul D. , Drinkwater Bruce W.
Published: August 2005

Two-dimensional (2D) phased arrays have the potential to significantly change the way in which engineering components in safety critical industries are inspected. In addition to enabling a three-dimensional (3D) volume of a component to be inspected from a single location, they could also be used in a C-scan configuration. The latter would enable any point in a component to be interrogated over a range of solid angles, allowing more accurate defect characterization and sizing. This paper describes the simulation and evaluation of grid, cross and circular 2D phased array element configurations. The aim of the cross and circle configurations is to increase the effective aperture for a given number of elements. Due to the multitude of possible array element configurations a model, based on Huygens’ principle, has been developed to allow analysis and comparison of candidate array designs. In addition to the element configuration, key issues such as element size, spacing, and frequency are discussed and quantitatively compared using the volume of the 3D point spread function (PSF) as a measurand. The results of this modeling indicate that, for a given number of elements, a circular array performs best and that the element spacing should be less than half a wavelength to avoid grating lobes. A prototype circular array has been built and initial results are presented. These show that a flat bottomed hole, half a wavelength in diameter, can be imaged. Furthermore, it is shown that the volume of the 3D reflection obtained experimentally from the end of the hole compares well with the volume of the 3D PSF predicted for the array at that point.
Microsatellite markers linked to 2 powdery mildew resistance genes introgressed fromTriticum carthlicumaccession PS5 into common wheat
Authors: Zhu Zhendong , Zhou Ronghua , Kong Xiuying , Dong Yuchen , Jia Jizeng
Published: August 2005

Two dominant powdery mildew resistance genes introduced from Triticum carthlicum accession PS5 to common wheat were identified and tagged using microsatellite markers. The gene designated PmPS5A was placed on wheat chromosome 2AL and linked to the microsatellite marker Xgwm356 at a genetic distance of 10.2 cM. Based on the information of its origin, chromosome location, and reactions to 5 powdery mildew isolates, this gene could be a member of the complex Pm4 locus. The 2nd gene designated PmPS5B was located on wheat chromosome 2BL with 3 microsatellite markers mapping proximally to the gene: Xwmc317 at 1.1 cM; Xgwm111 at 2.2 cM; and Xgwm382 at 4.0 cM; and 1 marker, Xgwm526, mapping distally to the gene at a distance of 18.1 cM. Since this gene showed no linkage to the other 2 known powdery mildew resistance genes on wheat chromosome 2B, Pm6 and Pm26, we believe it is a novel powdery mildew resistance gene and propose to designate this gene as Pm33.Key words: powdery mildew, resistance gene, microsatellite, Triticum carthlicum, wheat.
In vivo performance of a dual genetic marker,manA-gfp, in transgenic bentgrass
Authors: Fu Daolin , Xiao Yanmei , Muthukrishnan Subbaratnam , Liang George H
Published: August 2005

A dual-marker combination, manA-gfp, comprising 2 independent expression cassettes of genes encoding an Escherichia coli phosphomannose isomerase (PMI) and a synthetic green fluorescent protein (GFP), was incorporated into the binary vector pPZP201. Agrobacterium tumefaciens-mediated transfer was used to introduce the manA-gfp into the mature-seed derived calli of Agrostis stoloifera L. 'Crenshaw'. The putative transgenic bentgrass calli were screened in Murashige and Skoog medium containing 15 g mannose/L, in conjunction with a visual examination of the GFP expression with a fluorescence stereomicroscope. Calli with GFP fluorescence grew well on the mannose selection media. A total of 24 transgenic plants derived from a single piece of callus lobe were studied for the genomic integration, expression, and function of the transgene. Genomic integration of the dual markers manA and gfp was confirmed by Southern blotting analysis, and the expression of manA also was validated by using PMI-specific antiserum. The inheritance and expression of the dual marker, manA-gfp, was demonstrated in the T1generation. This study on the environmentally friendly markers further documented the feasibility of using alternative selection methods without using herbicide- or antibiotic-resistance markers.Key words: bentgrass, Agrobacterium tumefaciens-mediated transformation, chlorophenol red assay, phosphomannose isomerase (PMI).
Origin, structure, and behavior of a highly rearranged deletion chromosome 1BS-4 in wheat
Authors: Qi Lili , Friebe Bernd , Gill Bikram S
Published: August 2005

Wheat (Triticum aestivum L.) deletion (del) stocks are valuable tools for the physical mapping of molecular markers and genes to chromosome bins delineated by 2 adjacent deletion breakpoints. The wheat deletion stocks were produced by using gametocidal genes derived from related Aegilops species. Here, we report on the origin, structure, and behavior of a highly rearranged chromosome 1BS-4. The cytogenetic and molecular marker analyses suggest that 1BS-4 resulted from 2 breakpoints in the 1BS arm and 1 breakpoint in the 1BL arm. The distal segment from 1BS, except for a small deleted part, is translocated to the long arm. Cytologically, chromosome 1BS-4 is highly stable, but shows a unique meiotic pairing behavior. The short arm of 1BS-4 fails to pair with a normal 1BS arm because of lack of homology at the distal ends. The long arm of 1BS-4 only pairs with a normal 1BS arm within the distal region translocated from 1BS. Therefore, using the 1BS-4 deletion stock for physical mapping will result in the false allocation of molecular markers and genes proximal to the breakpoint of 1BS-4.Key words: Triticum aestivum, wheat, deletion–translocation, physical mapping.
A genetic and molecular profile of third chromosome centric heterochromatin inDrosophilamelanogaster
Authors: Fitzpatrick K A , Sinclair D A , Schulze S R , Syrzycka M , Honda B M
Published: August 2005

In this review, we combine the results of our published and unpublished work with the published results of other laboratories to provide an updated map of the centromeric heterochromatin of chromosome 3 in Drosophila melanogaster. To date, we can identify more than 20 genes (defined DNA sequences with well-characterized functions and (or) defined genetic complementation groups), including at least 16 essential loci. With the ongoing emergence of data from genetic, cytological, and genome sequencing studies, we anticipate continued, substantial progress towards understanding the function, structure, and evolution of centric heterochromatin.Key words: heterochromatin, Drosophila, cytogenetics, genomics.
Colour genes (R and Rc) for grain and coleoptile upregulate flavonoid biosynthesis genes in wheat
Authors: Himi Eiko , Nisar Ahmed , Noda Kazuhiko
Published: August 2005

Pigmentation of wheat grain and coleoptile is controlled by the R gene on chromosomes of the homoeologous group 3 and the Rc gene on chromosomes of the homoeologous group 7, respectively. Each of these genes is inherited monogenically. The pigment of grain has been suggested to be a derivative of catechin-tannin and that of coleoptile to be anthocyanin. These polyphenol compounds are known to be synthesized through the flavonoid biosynthesis pathway. We isolated 4 partial nucleotide sequences of the early flavonoid biosynthesis genes (CHS, CHI, F3H, and DFR) in wheat. The expression of these genes was examined in the developing grain of red-grained and white-grained wheat lines. CHS, CHI, F3H, and DFR were highly upregulated in the grain coat tissue of the red-grained lines, whereas there was no significant expression in the white-grained lines. These results indicate that the R gene is involved in the activation of the early flavonoid biosynthesis genes. As for coleoptile pigmentation, all 4 genes were expressed in the red coleoptile; however, DFR was not activated in the white coleoptile. The Rc gene appears to be involved in DFR expression. The possibility that wheat R and Rc genes might be transcription factors is discussed.Key words: flavonoid biosynthesis genes, R gene for grain color, Rc gene for coleoptile color, wheat.
A library ofSolanum lycopersicoidesintrogression lines in cultivated tomato
Authors: Canady Michael A , Meglic Vladimir , Chetelat Roger T
Published: August 2005

A set of introgression lines (ILs), containing individual chromosome segments from the wild nightshade Solanum lycopersicoides bred into the genetic background of cultivated tomato (Lycopersicon esculentum), has been developed. A primary group of 56 lines was selected for maximum representation of the S. lycopersicoides genome (~96% of the total map units), homozygosity, and a minimum number of introgressed segments per line. A secondary set of 34 lines provides increased map resolution in certain regions. Approximately 34% of the lines were sterile in the homozygous condition, but could be maintained by heterozygotes. To facilitate identification of segregating ILs, restriction fragment length polymorphism probes were converted to higher throughput cleaved amplified polymorphic sequence markers, which supplement allozyme and morphological loci. Strong segregation distortion was observed in F2progeny of heterozygous ILs, with an excess of L. esculentum alleles in most regions. For introgressions on distal chromosome 1L, a preferential transmission of S. lycopersicoides alleles was observed in the male germ line. Homozygous ILs generally yielded less seed from self pollination than corresponding heterozygotes, indicating that sterility effects were recessive. This IL library provides a novel resource for genetic studies of traits found in S. lycopersicoides.Key words: Lycopersicon esculentum, Solanum lycopersicum, Solanum lycopersicoides, segregation distortion, alien introgression.
Analyzing the potato abiotic stress transcriptome using expressed sequence tags
Authors: Rensink Willem , Hart Amy , Liu Jia , Ouyang Shu , Zismann Victoria , Buell C Robin
Published: August 2005

To further increase our understanding of responses in potato to abiotic stress and the potato transcriptome in general, we generated 20 756 expressed sequence tags (ESTs) from a cDNA library constructed by pooling mRNA from heat-, cold-, salt-, and drought-stressed potato leaves and roots. These ESTs were clustered and assembled into a collection of 5240 unique sequences with 3344 contigs and 1896 singleton ESTs. Assignment of gene ontology terms (GOSlim/Plant) to the sequences revealed that 8101 assignments could be made with a total of 3863 molecular function assignments. Alignment to a set of 78 825 ESTs from other potato cDNA libraries derived from root, leaf, stolon, tuber, germinating eye, and callus tissues revealed 1476 sequences unique to abiotic stressed potato leaf and root tissue. Sequences present within the 5240 sequence set had similarity to genes known to be involved in abiotic stress responses in other plant species such as transcription factors, stress response genes, and signal transduction processes. In addition, we identified a number of genes unique to the abiotic stress library with unknown function, providing new candidate genes for investigation of abiotic stress responses in potato.Key words: potato, Solanacaeae, abiotic stress.
Identification by genome scanning approach (GSA) of a microsatellite tightly associated with the apple scab resistance geneVm
Authors: Patocchi A , Walser M , Tartarini S , Broggini G A.L , Gennari F , Sansavini S , Gessler C
Published: August 2005

For all known major apple scab resistance genes except Vr, molecular markers have been published. However, the precise position of some of these genes, in the apple genome, remains to be identified. Knowledge about the relative position of apple scab resistance genes is necessary to preliminarily evaluate the probability of success of their pyramidization. Pyramidization of different resistance genes into the same genotype is a reliable way to create cultivars with durable apple scab resistance. Applying the genome scanning approach (GSA), we identified the linkage group of the scab resistance gene Vm, derived from Malus micromalus, and we found a new molecular marker tightly associated with the gene. The simple sequence repeat Hi07h02, previously mapped on linkage group 17, cosegregates with the Vm gene (no recombinants in the 95 plants tested). The already published sequence-characterized amplified region Vm marker OPB12687was found to be linked at about 5 cM from the resistance gene and, therefore, this marker also maps on linkage group 17 of apple. This is the first report of the discovery of a major apple scab resistance gene on linkage group 17. The advantages of using GSA for the identification of molecular markers for qualitative traits are discussed.Key words: Malus, Venturia inaequalis, mapping, simple sequence repeat.
Analysis of expressed sequence tags of the water flea Daphnia magna
Authors: Watanabe Hajime , Tatarazako Norihisa , Oda Shigeto , Nishide Hiroyo , Uchiyama Ikuo , Morita Masatoshi , Iguchi Taisen
Published: August 2005

To study gene expression in the water flea Daphnia magna we constructed a cDNA library and characterized the expressed sequence tags (ESTs) of 7210 clones. The EST sequences clustered into 2958 nonredundant groups. BLAST analyses of both protein and DNA databases showed that 1218 (41%) of the unique sequences shared significant similarities to known nucleotide or amino acid sequences, whereas the remaining 1740 (59%) showed no significant similarities to other genes. Clustering analysis revealed particularly high expression of genes related to ATP synthesis, structural proteins, and proteases. The cDNA clones and EST sequence information should be useful for future functional analysis of daphnid biology and investigation of the links between ecology and genomics.Key words: Daphnia magna, EST, classification, ATP synthesis.
Analysis of polyethylene pipe behaviour in horizontal directional drilling field tests
Authors: Polak Maria Anna
Published: August 2005

The paper presents results of both tests and analysis of polyethylene pipes installed using horizontal directional drilling (HDD). A total of seven full-scale HDD field installations were performed at the University of Waterloo. The test parameters were pull length, overcut ratio, quality of the drilling fluid, pipe diameter, and pipe material. The paper presents an overview of the testing methodology, instrumentation, and test specimens. The method for measuring strains along the length of the pipe is presented and discussed. Experimental results concentrate on pipe strains due to bending and axial deformations during HDD. Post-installation axial strains are also presented. A predictive model has been developed to analyze the pipe under various installation conditions. The model accounts for major mechanical contributions to the pulling force in HDD installations. The emphasis in this paper is on the comparison between theoretical and experimental results and the discussion of factors influencing pulling loads and strains in polyethylene pipes installed using HDD.Key words: horizontal directional drilling, polyethylene, pipe, analysis, tests, strain, pulling loads.
Measurements of void fraction and bubble properties on a stepped chute using a fiber-optic probe
Authors: EL-Kamash M K , Loewen M R , Rajaratnam N
Published: August 2005

This paper presents results from an experimental laboratory study conducted to investigate the use of a fiber-optic probe for measuring void fraction, bubble sizes, and bubble velocities in the highly aerated flow generated over a stepped chute. The accuracy of the measurements was verified by comparing the discharge measured by using a magnetic flow meter with the integrated discharge computed from the time averaged void fraction and bubble velocity measurements. At a sample rate of 20 kHz the average errors in the void fraction, bubble velocity, and bubble size were estimated to be 1%, 5%, and 6%, respectively. Increasing the data sample rate reduces the errors in the bubble size and velocity. It was concluded that this fiber-optic probe is well suited for making measurements of the void fraction, bubble sizes, and bubble velocities in a unidirectional two-phase flow.Key words: stepped chute, jet flow, air entrainment, fiber-optic probe, air bubble.
Nonlinear damage behaviour of concrete structures
Authors: Labadi Youcef , Hannachi Naceur Eddine
Published: August 2005

Concrete structures are becoming more and more sophisticated and submitted to severe conditions, for example: high stresses and temperatures, cyclic loadings, earthquakes, etc. It is therefore necessary to simulate correctly the behaviour and damage of such structures. However, the behaviour of this material is among the most complex ones: various phenomena are observed experimentally, such as, loss of stiffness, irreversible strains, stiffness recovery and dissymmetric behaviour to mention a few. If all these effects are taken into consideration, it would lead to models that use numerous parameters. In this paper, a framework for damage mechanics of concrete is presented and used to simulate the nonlinear behaviour of concrete using finite element method (FEM). A relatively simple isotropic damage model, containing essentially no adjustable parameters is shown to produce results in remarkably good agreement with experimental results. Indeed, the damage law requires only the fracture energy to be completely defined. A special form of damage surfaces is constructed to illustrate the application of the model. A new damage criterion, defined as an equivalent strain norm, is proposed to take into consideration the dissymmetric behaviour of concrete. To verify the FEM program including the model, the predicted deformations are compared with experimental results and results from other nonlinear constitutive models.Key words: elasticity, quasi-brittle materials, damage, cracking, nonlinear behaviour, concrete modelling, finite element, simulation.
Profit-point analysis: A tool for general contractors to measure and compare costs of management time expended on different subcontractors
Authors: Kim Yong-Woo , Ballard Glenn
Published: August 2005

One of the trends in construction today is the increasing use of specialty contractors. As a result, projects are becoming more complicated and fragmented, more coordination is required, and overhead costs of the general contractors are increasing relative to the direct costs. Better ways of controlling job-site overhead costs are needed. This paper presents profit-point analysis (PPA), a method for analyzing how indirect staff time of a general contractor is actually spent on a project. Profit points are imaginary points where a general contractor and subcontractors are interfaced. The PPA is a method of analysis on these points, which adapts activity-based costing from manufacturing to construction. This new method, illustrated through a case study, yields valuable information for managerial control; for example, the different amount of supplemental support from the general contractor required by different subcontractors.Key words: overhead costs, cost analysis, profit points, activity-based costing (ABC), management efficiency, evaluating specialty contractors.
Clear-water scour at bridge piers in fine and medium gravel beds
Authors: Raikar Rajkumar V , Dey Subhasish
Published: August 2005

An experimental investigation on scour at circular and square piers in uniform and non-uniform gravels (fine and medium sizes) under clear-water scour at limiting stability of gravels is presented. From the experimental results, it is observed that the equilibrium scour depth increases with decrease in gravel size. The variation of equilibrium scour depth with gravel sizes departures considerably from that with sand sizes. Consequently, the resulting sediment size factors for gravels, obtained from envelope curve fitting, are significantly different from the existing sediment size factor for sands. The influence of gravel gradation on scour depth is also prominent in non-uniform gravels. The time scales to represent the time variation of scour depth in uniform and non-uniform gravels are determined. For uniform gravels, the non-dimensional time scale increases with increase in pier Froude number and gravel size, whereas for non-uniform gravels, it decreases with increase in geometric standard deviation of particle size distribution of gravels.Key words: bridge pier, gravel beds, scour, erosion, sediment transport, open channel flow, hydraulic engineering.
Seismic response of concrete rectangular tanks for liquid containing structures
Authors: Chen J Z , Kianoush M R
Published: August 2005

In this paper, a procedure for computing hydrodynamic pressures in rectangular tanks is proposed. The procedure, which is referred to as the sequential method, considers the effect of the flexibility of the tank wall in determining the hydrodynamic pressures. In this study, only the impulsive response of the tank is considered. Based on a two-dimensional model of the tank wall, dynamic time-history analysis is carried out to study the effect of wall flexibility on the response. In the analysis, both a tall tank and a shallow tank are considered. The results of analysis are compared with those obtained based on current design practice codes and standards. The well-known Housner's model, which assumes that the mass of liquid is lumped on the wall based on rigid wall boundary condition in the calculation of hydrodynamic pressure, is widely used in practice. A comparison shows that in most cases, the lumped mass approach overestimates the base shear. The effect of wall flexibility on wall displacements, base shears, and moments are also discussed.Key words: reinforced concrete, liquid containing, rectangular tank, seismic, dynamic analysis, tank flexibility.
Organizing the genome: enhancers and insulators
Authors: Zhao Hui , Dean Ann
Published: August 2005

Enhancers can activate their target genes over large linear distances. Insulators can delimit the influence of an enhancer to an appropriate target. There are a number of intertwined mechanisms by which the regulatory functions of enhancers and insulators might be carried out at the level of the chromatin fiber. Recent evidence suggests that both enhancers and insulators participate in higher-order organization of chromatin in the nucleus and in localization of their regulated sequences to both subnuclear structures and compartments. Novel experimental approaches are helping to reveal the mechanisms underlying nuclear organization of developmentally regulated genes.Key words: enhancer, locus control region, insulator, chromatin, intergenic transcription, nucleus
Tilling the chromatin landscape: emerging methods for the discovery and profiling of protein–DNA interactions
Authors: Rodriguez Benjamin A.T , Huang Tim H.-M
Published: August 2005

Interactions between protein and DNA are essential for cellular function. The incremental process of developing global approaches to study chromatin began with the in vitro characterization of chromatin structural components and modifications of the versatile chromatin immunoprecipitation (ChIP) assay, capable of analyzing protein–DNA interactions in vivo. Among the emerging global approaches are ChIP cloning, ChIP display, differential chromatin scanning, ChIP–chip, DamID chromatin profiling, and chromatin array. These methods have been used to assess transcription-factor binding and (or) histone modification. This review describes these global methods and illustrates their potential in answering biological questions.Key words: ChIP, transcription factor binding, histone modification, ChIP display, differential chromatin scanning, ChIP-chip, DamID chromatin profiling, chromatin array.
Chromatin remodeling complexes: ATP-dependent machines in action
Authors: Johnson Cotteka N , Adkins Nicholas L , Georgel Philippe
Published: August 2005

Since the initial characterization of chromatin remodeling as an ATP-dependent process, many studies have given us insight into how nucleosome-remodeling complexes can affect various nuclear functions. However, the multistep DNA-histone remodeling process has not been completely elucidated. Although new studies are published on a nearly weekly basis, the nature and roles of interactions of the individual SWI/SNF- and ISWI-based remodeling complexes and DNA, core histones, and other chromatin-associated proteins are not fully understood. In addition, the potential changes associated with ATP recruitment and its subsequent hydrolysis have not been fully characterized. This review explores possible mechanisms by which chromatin-remodeling complexes are recruited to specific loci, use ATP hydrolysis to achieve actual remodeling through disruption of DNA-histone interactions, and are released from their chromatin template. We propose possible roles for ATP hydrolysis in a chromatin-release/target-scanning process that offer an alternative to or complement the often overlooked function of delivering the energy required for sliding or dislodging specific subsets of core histones.Key words: chromatin remodeling, SWI/SNF, ISWI, APT hydrolysis.
Cross-talking histones: implications for the regulation of gene expression and DNA repair
Authors: Wood Adam , Schneider Jessica , Shilatifard Ali
Published: August 2005

The regulation of chromatin structure is essential to life. In eukaryotic organisms, several classes of protein exist that can modify chromatin structure either through ATP-dependent remodeling or through the post-translational modification of histone proteins. A vast array of processes ranging from transcriptional regulation to DNA repair rely on these histone-modifying enzymes. In the last few years, enzymes involved in the post-translational modification of histone proteins have become a topic of intense interest. Our work and the work of several other laboratories has focused largely on understanding the biological role of the yeast histone methyltransferase COMPASS (complex of proteins associated with Set1) and its human homologue the MLL complex. The Set1-containing complex COMPASS acts as the sole histone H3 lysine 4 methyltransferase in Saccharomyces cerevisiae, and this methyl mark is important for transcriptional regulation and silencing at the telomeres and rDNA loci. Another histone methyltransferase, Dot1, methylates lysine 79 of histone H3 and is also essential for proper silencing of genes near telomeres, the rDNA loci, and the mating type loci. Employing our global biochemical screen GPS (global proteomic analysis of S. cerevisiae) we have been successful in identifying and characterizing several key downstream and upstream regulators of both COMPASS and Dot1 histone methyltransferase activity. This review details efforts made towards understanding the regulatory mechanisms and biological significance of COMPASS and Dot1p-mediated histone methylation.
How does the histone code work?
Authors: Cosgrove Michael S , Wolberger Cynthia
Published: August 2005

Patterns of histone post-translational modifications correlate with distinct chromosomal states that regulate access to DNA, leading to the histone-code hypothesis. However, it is not clear how modification of flexible histone tails leads to changes in nucleosome dynamics and, thus, chromatin structure. The recent discovery that, like the flexible histone tails, the structured globular domain of the nucleosome core particle is also extensively modified adds a new and exciting dimension to the histone-code hypothesis, and calls for the re-examination of current models for the epigenetic regulation of chromatin structure. Here, we review these findings and other recent studies that suggest the structured globular domain of the nucleosome core particle plays a key role regulating chromatin dynamics.Key words: histones, histone code, modifications, epigenetic, chromatin, nucleosome, dynamics, regulated nucleosome mobility, core, archaeal, combinatorial switch, histone octamer.
Maintenance and regulation of DNA methylation patterns in mammals
Authors: Chen Zhao-xia , Riggs Arthur D
Published: August 2005

Proper establishment and faithful maintenance of epigenetic information is crucial for the correct development of complex organisms. For mammals, it is now accepted that DNA methylation is an important mechanism for establishing stable heritable epigenetic marks. The distribution of methylation in the genome is not random, and patterns of methylated and unmethylated DNA are well regulated during normal development. The molecular mechanisms by which methylation patterns are established and maintained are complex and just beginning to be understood. In this review, we summarize recent progress in understanding the regulation of mammalian DNA methylation patterns, with an emphasis on the emerging roles of several protein and possible RNA factors. We also revisit the stochastic model of maintenance methylation and discuss its implications for epigenetic fidelity and gene regulation.Key words: Epigenetics, epigenetic fidelity, DNA methyltransferase, DNA demethylase, gene regulation.
Epigenetic silencing of theMGMTgene in cancer
Authors: Soejima Hidenobu , Zhao Wei , Mukai Tsunehiro
Published: August 2005

Silencing of the O6-methylguanine-DNA methyltransferase (MGMT) gene, a key to DNA repair, plays a critical role in the development of cancer. The gene product, functioning normally, removes a methyl group from mutagenic O6-methylguanine, which is produced by alkylating agents and can make a mismatched pair with thymine, leading to transition mutation through DNA replication. MGMT is epigenetically silenced in various human tumors. It is well known that DNA hypermethylation at the promoter CpG island plays a pivotal role in the epigenetic silencing of tumor suppressor genes. MGMT silencing, however, occurs without DNA hypermethylation in some cancer cells. Dimethylation of histone H3 lysine 9 and binding of methyl-CpG binding proteins are common and essential in MGMT-silenced cells. Silencing of MGMT has been shown to be a poor prognostic factor but a good predictive marker for chemotherapy when alkylating agents are used. In this review, we describe recent advances in understanding the silencing of MGMT and its role in carcinogenesis; epigenetic mechanisms; and clinical implications.Key words: DNA repair, cancer, DNA methylation, histone modification, MBD protein, heterochromatin.
Muscle LIM protein plays both structural and functional roles in skeletal muscle
Authors: Barash Ilona A. , Mathew Liby , Lahey Michele , Greaser Marion L. , Lieber Richard L.
Published: August 2005

Muscle LIM protein (MLP) has been suggested to be an important mediator of mechanical stress in cardiac tissue, but the role that it plays in skeletal muscle remains unclear. Previous studies have shown that it is dramatically upregulated in fast-to-slow fiber-type transformation and also after eccentric contraction (EC)-induced muscle injury. The functional consequences of this upregulation, if any, are unclear. In the present study, we have examined the skeletal muscle phenotype of MLP-knockout (MLPKO) mice in terms of their response to EC-induced muscle injuries. The data suggest that while the MLPKO mice recover completely after EC-induced injury, their torque production lags behind that of heterozygous littermates in the early stages of the recovery process. This lag is accompanied by decreased expression of the muscle regulatory factor MyoD, suggesting that MLP may influence gene expression. In addition, there is evidence of type I fiber atrophy and a shorter resting sarcomere length in the MLPKO mice, but no significant differences in fiber type distribution. In summary, MLP appears to play a subtle role in the maintenance of normal muscle characteristics and in the early events of the recovery process of skeletal muscle to injury, serving both structural and gene-regulatory roles.
Quantification of gap junction selectivity
Authors: Ek-Vitorín Jose F. , Burt Janis M.
Published: August 2005

Gap junctions, which are essential for functional coordination and homeostasis within tissues, permit the direct intercellular exchange of small molecules. The abundance and diversity of this exchange depends on the number and selectivity of the comprising channels and on the transjunctional gradient for and chemical character of the permeant molecules. Limited knowledge of functionally significant permeants and poor detectability of those few that are known have made it difficult to define channel selectivity. Presented herein is a multifaceted approach to the quantification of gap junction selectivity that includes determination of the rate constant for intercellular diffusion of a fluorescent probe (k2-DYE) and junctional conductance ( gj) for each junction studied, such that the selective permeability (k2-DYE/ gj) for dyes with differing chemical characteristics or junctions with differing connexin (Cx) compositions (or treatment conditions) can be compared. In addition, selective permeability can be correlated using single-channel conductance when this parameter is also measured. Our measurement strategy is capable of detecting 1) rate constants and selective permeabilities that differ across three orders of magnitude and 2) acute changes in that rate constant. Using this strategy, we have shown that 1) the selective permeability of Cx43 junctions to a small cationic dye varied across two orders of magnitude, consistent with the hypothesis that the various channel configurations adopted by Cx43 display different selective permeabilities; and 2) the selective permeability of Cx37 vs. Cx43 junctions was consistently and significantly lower.
N-methyl-d-glucamine and propidium dyes utilize different permeation pathways at rat P2X7 receptors
Authors: Jiang Lin-Hua , Rassendren Francois , Mackenzie Amanda , Zhang Yi-Hong , Surprenant Annmarie , North R. Alan
Published: August 2005

Activation of membrane P2X7 receptors by extracellular ATP [or its analog 2′,3′- O-(4-benzoylbenzoyl)-ATP] results in the opening within several milliseconds of an integral ion channel that is permeable to small cations. If the ATP application is maintained for several seconds, two further sequelae occur: there is a gradual increase in permeability to the larger cation N-methyl-d-glucamine and the cationic propidium dye quinolinium, 4-[(3-methyl-2(3 H)-benzoxazolylidene)methyl]-1-[3-(triethylammonio)propyl]diiodide (YO-PRO-1) enters the cell. The similarity in the time course of these two events has led to the widespread view that N-methyl-d-glucamine and YO-PRO-1 enter through a common permeation pathway, the “dilating” P2X7 receptor pore. Here we provide two independent lines of evidence against this view. We studied single human embryonic kidney cells expressing rat P2X7 receptors with patch-clamp recordings of membrane current and with fluorescence measurements of YO-PRO-1 uptake. First, we found that maintained application of the ATP analog did not cause any increase in N-methyl-d-glucamine permeability when the extracellular solution contained its normal sodium concentration, although YO-PRO-1 uptake was readily observed. Second, we deleted a cysteine-rich 18-amino acid segment in the intracellular juxtamembrane region of the P2X7 receptor. This mutated receptor showed normal YO-PRO-1 uptake but had no permeability to N-methyl-d-glucamine. Together, the clear differential effects of extracellular sodium ions or of mutation of the receptor strongly suggest that N-methyl-d-glucamine and YO-PRO-1 do not enter the cell by the same permeation pathway.
Calcium phosphate precipitation in the sarcoplasmic reticulum reduces action potential-mediated Ca2+ release in mammalian skeletal muscle
Authors: Dutka T. L. , Cole L. , Lamb G. D.
Published: August 2005

During vigorous exercise, Pi concentration levels within the cytoplasm of fast-twitch muscle fibers may reach ≥30 mM. Cytoplasmic Pi may enter the sarcoplasmic reticulum (SR) and bind to Ca2+ to form a precipitate (CaPi), thus reducing the amount of releasable Ca2+. Using mechanically skinned rat fast-twitch muscle fibers, which retain the normal action potential-mediated Ca2+ release mechanism, we investigated the consequences of Pi exposure on normal excitation-contraction coupling. The total amount of Ca2+ released from the SR by a combined caffeine/low-Mg2+ concentration stimulus was reduced by ∼20%, and the initial rate of force development slowed after 2-min exposure to 30 mM Pi (with or without the presence creatine phosphate). Peak (50 Hz) tetanic force was also reduced (by ∼25% and ∼45% after 10 and 30 mM Pi exposure, respectively). Tetanic force responses produced after 30 mM Pi exposure were nearly identical to those observed in the same fiber after depletion of total SR Ca2+ by ∼35%. Ca2+ content assays revealed that the total amount of Ca2+ in the SR was not detectably changed by exposure to 30 mM Pi, indicating that Ca2+ had not leaked from the SR but instead formed a precipitate with the Pi, reducing the amount of available Ca2+ for rapid release. These results suggest that CaPi precipitation that occurs within the SR could contribute to the failure of Ca2+ release observed in the later stages of metabolic muscle fatigue. They also demonstrate that the total amount of Ca2+ stored in the SR cannot drop substantially below the normal endogenous level without reducing tetanic force responses.
Na+-dependent HCO3− uptake into the rat choroid plexus epithelium is partially DIDS sensitive
Authors: Bouzinova Elena V. , Praetorius Jeppe , Virkki Leila V. , Nielsen Søren , Boron Walter F. , Aalkjaer Christian
Published: August 2005

Several studies suggest the involvement of Na+ and HCO3− transport in the formation of cerebrospinal fluid. Two Na+-dependent HCO3− transporters were recently localized to the epithelial cells of the rat choroid plexus (NBCn1 and NCBE), and the mRNA for a third protein was also detected (NBCe2) (Praetorius J, Nejsum LN, and Nielsen S. Am J Physiol Cell Physiol 286: C601–C610, 2004). Our goal was to immunolocalize the NBCe2 to the choroid plexus by immunohistochemistry and immunogold electronmicroscopy and to functionally characterize the bicarbonate transport in the isolated rat choroid plexus by measurements of intracellular pH (pHi) using a dual-excitation wavelength pH-sensitive dye (BCECF). Both antisera derived from COOH-terminal and NH2-terminal NBCe2 peptides localized NBCe2 to the brush-border membrane domain of choroid plexus epithelial cells. Steady-state pHi in choroidal cells increased from 7.03 ± 0.02 to 7.38 ± 0.02 ( n = 41) after addition of CO2/HCO3− into the bath solution. This increase was Na+ dependent and inhibited by the Cl− and HCO3− transport inhibitor DIDS (200 μM). This suggests the presence of Na+-dependent, partially DIDS-sensitive HCO3− uptake. The pHi recovery after acid loading revealed an initial Na+ and HCO3−-dependent net base flux of 0.828 ± 0.116 mM/s ( n = 8). The initial flux in the presence of CO2/HCO3− was unaffected by DIDS. Our data support the existence of both DIDS-sensitive and -insensitive Na+- and HCO3−-dependent base loader uptake into the rat choroid plexus epithelial cells. This is consistent with the localization of the three base transporters NBCn1, Na+-driven Cl− bicarbonate exchanger, and NBCe2 in this tissue.
Differential effects of superoxide on luminal and basolateral Na+/H+ exchange in the thick ascending limb
Authors: Juncos Ramiro , Hong Nancy J. , Garvin Jeffrey L.
Published: August 2005

Superoxide (O2−) increases Na+ reabsorption in the thick ascending limb (THAL) by enhancing Na/K/2Cl cotransport. However, the effects of O2− on other THAL transporters, such as Na+/H+ exchangers, are unknown. We hypothesized that O2− stimulates Na+/H+ exchange in the THAL. We assessed total Na+/H+ exchange activity by measuring recovery of intracellular pH (pHi) after acid loading in isolated perfused THALs before and after adding xanthine oxidase (XO) and hypoxanthine (HX). We found that XO and HX decreased total pHi recovery rate from 0.26 ± 0.05 to 0.21 ± 0.04 pH units/min ( P < 0.05), and this net inhibition decreased steady-state pHi from 7.52 to 7.37. Because THALs have different Na+/H+ exchanger isoforms on the luminal and basolateral membrane, we tested the effects of xanthine oxidase and hypoxanthine on luminal and basolateral Na+/H+ exchange by adding dimethylamiloride to either the bath or lumen. Xanthine oxidase and hypoxanthine increased luminal Na+/H+ exchange from 3.5 ± 0.8 to 6.7 ± 1.4 pmol·min−1·mm−1 ( P < 0.01) but decreased basolateral Na+/H+ exchange from 10.8 ± 1.8 to 6.8 ± 1.1 pmol·min−1·mm−1 ( P < 0.007). To ascertain whether these effects were caused by O2− or H2O2, we examined the ability of tempol, a superoxide dismutase mimetic, to block these effects. In the presence of tempol, xanthine oxidase and hypoxanthine had no effect on luminal or basolateral Na+/H+ exchange. We conclude that O2− inhibits basolateral and stimulates luminal Na+/H+ exchangers, perhaps because different isoforms are expressed on each membrane. Inhibition of basolateral Na+/H+ exchange may enhance stimulation of luminal Na+/H+ exchange by providing additional protons to be extruded across the luminal membrane. Together, the effects of O2− on Na+/H+ exchange may increase net HCO3− reabsorption by the THAL.
Suppression of Ih Contributes to Propofol-Induced Inhibition of Mouse Cortical Pyramidal Neurons
Authors: Chen Xiangdong , Shu Shaofang , Bayliss Douglas A.
Published: August 2005

The contributions of the hyperpolarization-activated current, Ih, to generation of rhythmic activities are well described for various central neurons, particularly in thalamocortical circuits. In the present study, we investigated effects of a general anesthetic, propofol, on native Ih in neurons of thalamus and cortex and on the corresponding cloned HCN channel subunits. Whole cell voltage-clamp recordings from mouse brain slices identified neuronal Ih currents with fast activation kinetics in neocortical pyramidal neurons and with slower kinetics in thalamocortical relay cells. Propofol inhibited the fast-activating Ih in cortical neurons at a clinically relevant concentration (5 μM); inhibition of Ih involved a hyperpolarizing shift in half-activation voltage (Δ V1/2 approximately −9 mV) and a decrease in maximal available current (∼36% inhibition, measured at −120 mV). With the slower form of Ih expressed in thalamocortical neurons, propofol had no effect on current activation or amplitude. In heterologous expression systems, 5 μM propofol caused a large shift in V1/2 and decrease in current amplitude in homomeric HCN1 and linked heteromeric HCN1–HCN2 channels, both of which activate with fast kinetics but did not affect V1/2 or current amplitude of slowly activating homomeric HCN2 channels. With GABAA and glycine receptor channels blocked, propofol caused membrane hyperpolarization and suppressed action potential discharge in cortical neurons; these effects were occluded by the Ih blocker, ZD-7288. In summary, these data indicate that propofol selectively inhibits HCN channels containing HCN1 subunits, such as those that mediate Ih in cortical pyramidal neurons—and they suggest that anesthetic actions of propofol may involve inhibition of cortical neurons and perhaps other HCN1-expressing cells.
A Role for Hip Position in Initiating the Swing-to-Stance Transition in Walking Cats
Authors: McVea D. A. , Donelan J. M. , Tachibana A. , Pearson K. G.
Published: August 2005

In this investigation, we obtained data that support the hypothesis that afferent signals associated with hip flexion play a role in initiating the swing-to-stance transition of the hind legs in walking cats. Direct evidence came from observations in walking decerebrate cats. Assisting the flexion of the hip joint during swing advanced the onset of activity in ankle extensor muscles, and this advance was strongly correlated with a reduction in the duration of hip flexor muscle activity. The hip angle at the time of onset of the flexion to extension transition was similar during assisted and unassisted steps. Additional evidence for the hypothesis that sensory signals related to hip flexion are important in regulating the swing-to-stance transition came from four normal animals trained to walk in a variety of situations designed to alter the coordination of movements at the hip, knee, and ankle joints during the swing phase. Although there were exceptions in some tasks and preparations, the angle of the hip joint at the time of onset of extensor activity was generally less variable than that of the knee and ankle joints. We also found no clear relationships between the angle of the limb and body axes, or the length of the limb axis, and the time of onset of extensor activity. Finally, there were no indications that the stretching of ankle extensor muscles during swing was a factor in regulating the transition from swing-to-stance.
Role of Motor and Visual Experience During Development of Bipedal Locomotion in Chicks
Authors: Muir Gillian D. , Gowri K.S.V.
Published: August 2005

The purpose of this research was to investigate the role of motor and visual experience during the development of locomotion in chicks. We have previously demonstrated that when locomotor activity is restricted immediately posthatching, chicks walk with shorter stride lengths and attenuated head bobbing movements. Head bobbing is an optokinetic response in birds, driven by the movement of the visual world across the retina (i.e., optic flow). During locomotion, optic flow is generated by forward translation, and we have shown that the magnitude of head bobbing movements and stride lengths are moderately correlated in walking chicks. In the present study, we investigated this relationship more closely by examining whether imposed changes in stride length could affect head excursions during head bobbing. We manipulated stride length by hobbling chicks immediately after hatching and subsequently quantified kinematic parameters, including step timing and head excursions, during walking. Imposition of shorter stride lengths induced chicks to take more frequent steps, spend less time in contact with the ground, and shortened head excursions during head bobbing. Nevertheless, the developmental changes in head excursions were not fully accounted for by altered stride lengths, so in a separate experiment, we investigated whether the development of head bobbing relies on the normal experience of optic flow. We raised chicks under stroboscopic illumination to eliminate chicks' experience of optic flow but found that this did not significantly alter head bobbing. These results are discussed along with related findings in other species and the possible neural and biomechanical constraints underlying development of walking and head bobbing in birds.
Chromosomics
Authors: Claussen U.
Published: August 2005

The term “chromosomics” is introduced to draw attention to the three-dimensional morphological changes in chromosomes that are essential elements in gene regulation. Chromosomics deals with the plasticity of chromosomes in relation to the three-dimensional positions of genes, which affect cell function in a developmental and tissue-specific manner during the cell cycle. It also deals with species-specific differences in the architecture of chromosomes, which has been overlooked in the past. Chromosomics includes research into chromatin-modification-mediated changes in the architecture of chromosomes, which may influence the functions and life-spans of cells, tissues, organs and individuals. It also addresses the occurrence and prevalence of chromosomal gaps and breaks.