bluuebunny/crossref_metadata_2025 huggingface dataset:
A combinatorial role for NFAT5 in both myoblast migration and differentiation during skeletal muscle myogenesis
Authors: O'Connor Roddy S. , Mills Stephen T. , Jones Kristen A. , Ho Steffan N. , Pavlath Grace K.
Published: December 2006

Skeletal muscle regeneration depends on myoblast migration, differentiation and myofiber formation. Isoforms of the nuclear factor of activated T cells (NFAT) family of transcription factors display nonredundant roles in skeletal muscle. NFAT5, a new isoform of NFAT, displays many differences from NFATc1-c4. Here, we examine the role of NFAT5 in myogenesis. NFAT5+/- mice displayed a defect in muscle regeneration with fewer myofibers formed at early times after injury. NFAT5 has a muscle-intrinsic function because inhibition of NFAT5 transcriptional activity caused both a migratory and differentiation defect in cultured myoblasts. We identified Cyr61 as a target of NFAT5 signaling in skeletal muscle cells. Addition of Cyr61 to cells expressing inhibitory forms of NFAT5 rescued the migratory phenotype. These results demonstrate a role for NFAT5 in skeletal muscle cell migration and differentiation. Furthermore, as cell-cell interactions are crucial for myoblast differentiation, these data suggest that myoblast migration and differentiation are coupled and that NFAT5 is a key regulator.
Hyaluronan inhibits osteoclast differentiation via Toll-like receptor 4
Authors: Chang Eun-Ju , Kim Hyon Jong , Ha Jeongim , Kim Hyung Joon , Ryu Jiyoon , Park Kwang-Hyun , Kim Uh-Hyun , Lee Zang Hee , Kim Hyun-Man , Fisher David E. , Kim Hong-Hee
Published: December 2006

The differentiation of osteoclasts, cells specialized for bone resorption, is governed by two key factors, macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor κB ligand (RANKL). The extracellular matrix (ECM) is an important factor influencing cell fate. To date, little investigation on the relationship between ECM components and osteoclast differentiation has been documented. In this study, we uncovered a potent anti-osteoclastogenic effect of hyaluronan (HA), an ECM component present in bone marrow and soft connective tissues, in primary mouse and human osteoclast precursor cell cultures. The anti-osteoclastogenic function of HA was dependent on Toll-like receptor 4 (TLR4) but not on CD44. HA inhibited M-CSF-dependent signaling pathways involving Rac, reactive oxygen species and mitogen-activated protein kinases, resulting in suppression of transcription factors AP-1 and MITF that control RANK expression. Furthermore, in an in vivo mouse model of calvarial bone resorption assays HA reduced RANKL-induced bone erosion and osteoclastogenesis. Our results clearly show that HA inhibits osteoclast differentiation through TLR4 by interfering with M-CSF signaling, and point that the interaction between ECM components and innate immune receptors can play an important role in the regulation of bone metabolism.
Universality and Specificity of Polymer Gels Viewed by Scattering Methods
Authors: Shibayama Mitsuhiro
Published: December 2006

Concentration fluctuations in polymer gels are composed of dynamic thermal fluctuations and frozen inhomogeneities. Particularly, the latter is a characteristic of gels and is due to cross-linking. The frozen inhomogeneities manifest unique properties in polymer gels. Speckle patterns are commonly observed in gels. The time-correlation function of the scattering intensity entails a power-law behavior at the sol–gel transition. The appearance of speckle patterns is related to the nonergodicity of gels, and the power-law behavior corresponds to the self-similarity of clusters at the gelation threshold. In this review, it will be demonstrated that light scattering is one of the most powerful tools for structure characterization of polymer gels. Then, the universality and specificity of polymer gels will be examined for various types of gels, including physical gels, composite gels, and gelators.
Briaexcavatins G and H, Two New Briaranes from the Octocoral Briareum excavatum
Authors: Chen Yu-Pei , Wu Shwu-Li , Su Jui-Hsin , Lin Mei-Ru , Hu Wan-Ping , Hwang Tsong-Long , Sheu Jyh-Horng , Fan Tung-Yung , Fang Lee-Shing , Sung Ping-Jyun
Published: December 2006

Two new briarane-related diterpenoids, designated as briaexcavatins G (1) and H (2), were isolated from the octocoral Briareum excavatum, collected off southern Taiwan coast. The structures, including the absolute stereochemistry of these two new metabolites, were established by spectroscopy and chemical conversion. The configuration of briarane 2 was further supported by molecular mechanics calculations. Briarane 1 showed weak inhibitory effects on human neutrophil elastase release.
Efficient Monte Carlo and Quasi–Monte Carlo Option Pricing Under the Variance Gamma Model
Authors: Avramidis Athanassios N. , L’Ecuyer Pierre
Published: December 2006

We develop and study efficient Monte Carlo algorithms for pricing path-dependent options with the variance gamma model. The key ingredient is difference-of-gamma bridge sampling, based on the representation of a variance gamma process as the difference of two increasing gamma processes. For typical payoffs, we obtain a pair of estimators (named low and high) with expectations that (1) are monotone along any such bridge sampler, and (2) contain the continuous-time price. These estimators provide pathwise bounds on unbiased estimators that would be more expensive (infinitely expensive in some situations) to compute. By using these bounds with extrapolation techniques, we obtain significant efficiency improvements by work reduction. We then combine the gamma bridge sampling with randomized quasi–Monte Carlo to reduce the variance and thus further improve the efficiency. We illustrate the large efficiency improvements on numerical examples for Asian, lookback, and barrier options.
Common Method Variance in IS Research: A Comparison of Alternative Approaches and a Reanalysis of Past Research
Authors: Malhotra Naresh K. , Kim Sung S. , Patil Ashutosh
Published: December 2006

Despite recurring concerns about common method variance (CMV) in survey research, the information systems (IS) community remains largely uncertain of the extent of such potential biases. To address this uncertainty, this paper attempts to systematically examine the impact of CMV on the inferences drawn from survey research in the IS area. First, we describe the available approaches for assessing CMV and conduct an empirical study to compare them. From an actual survey involving 227 respondents, we find that although CMV is present in the research areas examined, such biases are not substantial. The results also suggest that few differences exist between the relatively new marker-variable technique and other well-established conventional tools in terms of their ability to detect CMV. Accordingly, the marker-variable technique was employed to infer the effect of CMV on correlations from previously published studies. Our findings, based on the reanalysis of 216 correlations, suggest that the inflated correlation caused by CMV may be expected to be on the order of 0.10 or less, and most of the originally significant correlations remain significant even after controlling for CMV. Finally, by extending the marker-variable technique, we examined the effect of CMV on structural relationships in past literature. Our reanalysis reveals that contrary to the concerns of some skeptics, CMV-adjusted structural relationships not only remain largely significant but also are not statistically differentiable from uncorrected estimates. In summary, this comprehensive and systematic analysis offers initial evidence that (1) the marker-variable technique can serve as a convenient, yet effective, tool for accounting for CMV, and (2) common method biases in the IS domain are not as serious as those found in other disciplines.
Measurement of late-night salivary cortisol with an automated immunoassay system
Authors: Vogeser Michael , Durner Jürgen , Seliger Ewald , Auernhammer Christoph
Published: December 2006

Clin Chem Lab Med 2006;44:1441–5.
Discrimination Training Alters Object Representations in Human Extrastriate Cortex
Authors: Op de Beeck Hans P. , Baker Chris I. , DiCarlo James J. , Kanwisher Nancy G.
Published: December 2006

Visual object recognition relies critically on learning. However, little is known about the effect of object learning in human visual cortex, and in particular how the spatial distribution of training effects relates to the distribution of object and face selectivity across the cortex before training. We scanned human subjects with high-resolution functional magnetic resonance imaging (fMRI) while they viewed novel object classes, both before and after extensive training to discriminate between exemplars within one of these object classes. Training increased the strength of the response in visual cortex to trained objects compared with untrained objects. However, training did not simply induce a uniform increase in the response to trained objects: the magnitude of this training effect varied substantially across subregions of extrastriate cortex, with some showing a twofold increase in response to trained objects and others (including the right fusiform face area) showing no significant effect of training. Furthermore, the spatial distribution of training effects could not be predicted from the spatial distribution of either pretrained responses or face selectivity. Instead, training changed the spatial distribution of activity across the cortex. These findings support a dynamic view of the ventral visual pathway in which the cortical representation of an object category is continuously modulated by experience.
Genetic Analysis of Mint/X11 Proteins: Essential Presynaptic Functions of a Neuronal Adaptor Protein Family
Authors: Ho Angela , Morishita Wade , Atasoy Deniz , Liu Xinran , Tabuchi Katsuhiko , Hammer Robert E. , Malenka Robert C. , Südhof Thomas C.
Published: December 2006

Mints/X11s are adaptor proteins composed of three isoforms: neuron-specific Mints 1 and 2, and the ubiquitously expressed Mint 3. We have now analyzed constitutive and conditional knock-out mice for all three Mints/X11s. We found that ∼80% of mice lacking both neuron-specific Mint isoforms (Mints 1 and 2) die at birth, whereas mice lacking any other combination of Mint isoforms survive normally. The ∼20% surviving Mint 1/2 double knock-out mice exhibit a decrease in weight and deficits in motor behaviors. Hippocampal slice electrophysiology uncovered a decline in spontaneous neurotransmitter release, lowered synaptic strength, and enhanced paired-pulse facilitation in Mint-deficient mice, suggesting a decreased presynaptic release probability. Acute ablation of Mint expression in cultured neurons from conditional Mint 1/2/3 triple knock-in mice also revealed a decline in spontaneous release, confirming that deletion of Mints impair presynaptic function. Quantitation of synaptic proteins showed that acute deletion of Mints caused a selective increase in Munc18-1 and Fe65 proteins, and overexpression of Munc18-1 in wild-type neurons also produced a decrease in spontaneous release, suggesting that the interaction of Mints with Munc18-1 may contribute to the presynaptic phenotype observed in Mint-deficient mice. Our studies thus indicate that Mints are important regulators of presynaptic neurotransmitter release that are essential for mouse survival.
A Physiologically Plausible Model of Action Selection and Oscillatory Activity in the Basal Ganglia
Authors: Humphries Mark D. , Stewart Robert D. , Gurney Kevin N.
Published: December 2006

The basal ganglia (BG) have long been implicated in both motor function and dysfunction. It has been proposed that the BG form a centralized action selection circuit, resolving conflict between multiple neural systems competing for access to the final common motor pathway. We present a new spiking neuron model of the BG circuitry to test this proposal, incorporating all major features and many physiologically plausible details. We include the following: effects of dopamine in the subthalamic nucleus (STN) and globus pallidus (GP), transmission delays between neurons, and specific distributions of synaptic inputs over dendrites. All main parameters were derived from experimental studies. We find that the BG circuitry supports motor program selection and switching, which deteriorates under dopamine-depleted and dopamine-excessive conditions in a manner consistent with some pathologies associated with those dopamine states. We also validated the model against data describing oscillatory properties of BG. We find that the same model displayed detailed features of both γ-band (30–80 Hz) and slow (∼1 Hz) oscillatory phenomena reported by Brown et al. (2002) and Magill et al. (2001), respectively. Only the parameters required to mimic experimental conditions (e.g., anesthetic) or manipulations (e.g., lesions) were changed. From the results, we derive the following novel predictions about the STN–GP feedback loop: (1) the loop is functionally decoupled by tonic dopamine under normal conditions and recoupled by dopamine depletion; (2) the loop does not show pacemaking activity under normal conditionsin vivo(but does after combined dopamine depletion and cortical lesion); (3) the loop has a resonant frequency in the γ-band.
The Voltage-Gated Sodium Channel Nav1.9 Is an Effector of Peripheral Inflammatory Pain Hypersensitivity
Authors: Amaya Fumimasa , Wang Haibin , Costigan Michael , Allchorne Andrew J. , Hatcher Jon P. , Egerton Julie , Stean Tania , Morisset Valerie , Grose David , Gunthorpe Martin J. , Chessell Iain P. , Tate Simon , Green Paula J. , Woolf Clifford J.
Published: December 2006

We used a mouse with deletion of exons 4, 5, and 6 of the SCN11A (sodium channel, voltage-gated, type XI, α) gene that encodes the voltage-gated sodium channel Nav1.9 to assess its contribution to pain. Nav1.9 is present in nociceptor sensory neurons that express TRPV1, bradykinin B2, and purinergic P2X3receptors. InNav1.9−/−mice, the non-inactivating persistent tetrodotoxin-resistant sodium TTXr-Per current is absent, whereas TTXr-Slow is unchanged. TTXs currents are unaffected by the mutation of Nav1.9. Pain hypersensitivity elicited by intraplantar administration of prostaglandin E2, bradykinin, interleukin-1β, capsaicin, and P2X3and P2Y receptor agonists, but not NGF, is either reduced or absent inNav1.9−/−mice, whereas basal thermal and mechanical pain sensitivity is unchanged. Thermal, but not mechanical, hypersensitivity produced by peripheral inflammation (intraplanatar complete Freund's adjuvant) is substantially diminished in the null allele mutant mice, whereas hypersensitivity in two neuropathic pain models is unchanged in theNav1.9−/−mice. Nav1.9 is, we conclude, an effector of the hypersensitivity produced by multiple inflammatory mediators on nociceptor peripheral terminals and therefore plays a key role in mediating peripheral sensitization.
Translational Control via the Mammalian Target of Rapamycin Pathway Is Critical for the Formation and Stability of Long-Term Fear Memory in Amygdala Neurons
Authors: Parsons Ryan G. , Gafford Georgette M. , Helmstetter Fred J.
Published: December 2006

The mammalian target of rapamycin kinase (mTOR) regulates protein synthesis in neurons at the translational level through phosphorylation of several intracellular targets. Recent work in invertebrates indicates that mTOR-dependent translational control may be critical for the induction and maintenance of activity-dependent synaptic plasticity underlying memory formation. Here, we report that training rats in a simple fear conditioning procedure evokes a time-dependent increase in the phosphorylation of p70s6 kinase, a major direct downstream target of mTOR. When the activation of mTOR was prevented by posttraining injection of rapamycin into the amygdala, formation of the memory and the increase in p70s6 kinase phosphorylation was attenuated. Furthermore, when rapamycin was applied to the amygdala after the recall of a previously stored fear memory, subsequent retention was disrupted, indicating that local translational control at active synapses is required for the stability as well as the formation of long-term memory in this system.
The Linear Computational Algorithm of Cerebellar Purkinje Cells
Authors: Walter Joy T. , Khodakhah Kamran
Published: December 2006

The orchestration of simple motor tasks by the cerebellum results in coordinated movement and the maintenance of balance. The cerebellum integrates sensory and cortical information to generate the signals required for the coordinated execution of simple motor tasks. These signals originate in the firing rate of Purkinje cells, each of which integrates sensory and cortical information conveyed by granule cell synaptic inputs. Given the importance of the granule cell input–Purkinje cell output function for cerebellar computation, this algorithm was determined. Using several stimulation paradigms, including those that mimicked patterns of granule cell activity similar to those observedin vivo, we quantified the poststimulus maximum firing rate and number of extra spikes in response to granule cell synaptic input. Both of these parameters linearly encoded the strength of synaptic input when inhibitory synaptic transmission was blocked. This linear algorithm was independent of the location or temporal pattern of synaptic input. With inhibitory synaptic transmission intact, the maximum firing rate, but not the number of extra spikes, encoded the strength of granule cell synaptic input. Furthermore, the maximum firing rate of Purkinje cells linearly encoded the strength of synaptic input whether or not the activation of granule cells resulted in a pause in Purkinje cell firing. On the basis of the data presented, we propose that Purkinje cells encode the strength of granule cell synaptic input in their maximum firing rate with a linear algorithm.
Mutual Responses in the Midst of an Era: Aaron Copland's The Tender Land and Leonard Bernstein's Candide
Authors: CRIST ELIZABETH B.
Published: December 2006

Aaron Copland's The Tender Land (1954) and Leonard Bernstein's Candide (1956) both comment sharply on McCarthyism, but their broader political implications are less clear. While the story of each may be read in terms of postwar liberalism, a position itself informed by anticommunist ideology, two crucial ensembles reflect a decidedly leftist perspective. “The Promise of Living,” which ends act 1 of The Tender Land, and “Make Our Garden Grow,” the finale of Candide, celebrate a grand, utopian ideal—the politically outmoded, progessive ideal of community as a collective forged through common experience and shared labor—in musical terms that evoke the era of the Popular Front.

Despite their similarities, however, “The Promise of Living” and “Make Our Garden Grow” arrive at structurally distinct points in their perspective works and announce different dramatic as well as political ends. Ultimately one community comes together while the other falls apart, and these divergent trajectories speak to the experiences of the composers themselves as representatives of separate political generations. Using achival research, cultural history, and music analysis, I argue that at these moments The Tender Land and Candide reject the conservative and liberal anticommunism of their mutual Cold War context to remember or imagine the possibilities of a vital Left.
Homeologous Recombination Plays a Major Role in Chromosome Rearrangements That Occur During Meiosis of Brassica napus Haploids
Authors: Nicolas Stéphane D , Mignon Guillaume Le , Eber Frédérique , Coriton Olivier , Monod Hervé , Clouet Vanessa , Huteau Virginie , Lostanlen Antoine , Delourme Régine , Chalhoub Boulos , Ryder Carol D , Chèvre Anne Marie , Jenczewski Eric
Published: December 2006

Chromosomal rearrangements can be triggered by recombination between distinct but related regions. Brassica napus (AACC; 2n = 38) is a recent allopolyploid species whose progenitor genomes are widely replicated. In this article, we analyze the extent to which chromosomal rearrangements originate from homeologous recombination during meiosis of haploid B. napus (n = 19) by genotyping progenies of haploid × euploid B. napus with molecular markers. Our study focuses on three pairs of homeologous regions selected for their differing levels of divergence (N1/N11, N3/N13, and N9/N18). We show that a high number of chromosomal rearrangements occur during meiosis of B. napus haploid and are transmitted by first division restitution (FDR)-like unreduced gametes to their progeny; half of the progeny of Darmor-bzh haploids display duplications and/or losses in the chromosomal regions being studied. We demonstrate that half of these rearrangements are due to recombination between regions of primary homeology, which represents a 10- to 100-fold increase compared to the frequency of homeologous recombination measured in euploid lines. Some of the other rearrangements certainly result from recombination between paralogous regions because we observed an average of one to two autosyndetic A–A and/or C–C bivalents at metaphase I of the B. napus haploid. These results are discussed in the context of genome evolution of B. napus.
Mitochondrial Genome Sequence Evolution in Chlamydomonas
Authors: Popescu Cristina E , Lee Robert W
Published: December 2006

The mitochondrial genomes of the Chlorophyta exhibit significant diversity with respect to gene content and genome compactness; however, quantitative data on the rates of nucleotide substitution in mitochondrial DNA, which might help explain the origin of this diversity, are lacking. To gain insight into the evolutionary forces responsible for mitochondrial genome diversification, we sequenced to near completion the mitochondrial genome of the chlorophyte Chlamydomonas incerta, estimated the evolutionary divergence between Chlamydomonas reinhardtii and C. incerta mitochondrial protein-coding genes and rRNA-coding regions, and compared the relative evolutionary rates in mitochondrial and nuclear genes. Synonymous and nonsynonymous substitution rates do not differ significantly between the mitochondrial and nuclear protein-coding genes. The mitochondrial rRNA-coding regions, however, are evolving much faster than their nuclear counterparts, and this difference might be explained by relaxed functional constraints on the mitochondrial translational apparatus due to the small number of proteins synthesized in Chlamydomonas mitochondria. Substitution rates at synonymous sites in a nonstandard mitochondrial gene (rtl) and at intronic and synonymous sites in nuclear genes expressed at low levels suggest that the mutation rate is similar in these two genetic compartments. Potential evolutionary forces shaping mitochondrial genome evolution in Chlamydomonas are discussed.
Haplotype Probabilities for Multiple-Strain Recombinant Inbred Lines
Authors: Teuscher Friedrich , Broman Karl W
Published: December 2006

Recombinant inbred lines (RIL) derived from multiple inbred strains can serve as a powerful resource for the genetic dissection of complex traits. The use of such multiple-strain RIL requires a detailed knowledge of the haplotype structure in such lines. Broman (2005) derived the two- and three-point haplotype probabilities for 2n-way RIL; the former required hefty computation to infer the symbolic results, and the latter were strictly numerical. We describe a simpler approach for the calculation of these probabilities, which allowed us to derive the symbolic form of the three-point haplotype probabilities. We also extend the two-point results for the case of additional generations of intermating, including the case of 2n-way intermated recombinant inbred populations (IRIP).
Functional Analysis of Gene Duplications in Saccharomyces cerevisiae
Authors: Guan Yuanfang , Dunham Maitreya J , Troyanskaya Olga G
Published: December 2006

Gene duplication can occur on two scales: whole-genome duplications (WGD) and smaller-scale duplications (SSD) involving individual genes or genomic segments. Duplication may result in functionally redundant genes or diverge in function through neofunctionalization or subfunctionalization. The effect of duplication scale on functional evolution has not yet been explored, probably due to the lack of global knowledge of protein function and different times of duplication events. To address this question, we used integrated Bayesian analysis of diverse functional genomic data to accurately evaluate the extent of functional similarity and divergence between paralogs on a global scale. We found that paralogs resulting from the whole-genome duplication are more likely to share interaction partners and biological functions than smaller-scale duplicates, independent of sequence similarity. In addition, WGD paralogs show lower frequency of essential genes and higher synthetic lethality rate, but instead diverge more in expression pattern and upstream regulatory region. Thus, our analysis demonstrates that WGD paralogs generally have similar compensatory functions but diverging expression patterns, suggesting a potential of distinct evolutionary scenarios for paralogs that arose through different duplication mechanisms. Furthermore, by identifying these functional disparities between the two types of duplicates, we reconcile previous disputes on the relationship between sequence divergence and expression divergence or essentiality.
The Evolutionary History of Drosophila buzzatii. XXXVI. Molecular Structural Analysis of Osvaldo Retrotransposon Insertions in Colonizing Populations Unveils Drift Effects in Founder Events
Authors: García Guerreiro María Pilar , Fontdevila Antonio
Published: December 2006

Previous work on transposable element distribution in colonizing populations of Drosophila buzzatii revealed a high frequency of occupancy in several chromosomal sites. Two explanatory hypotheses were advanced: the founder hypothesis, by which founder genetic drift was responsible, and the unstable hypothesis that assigns this unusual distribution to bursts of transposition toward some chromosomal sites. Here, we study the molecular structure of three euchromatic Osvaldo clones isolated from sites occupied at high (A4 and B9) and low frequency (B4) in colonizing populations, to test these hypotheses. Large insertions, duplications, and indels in the Osvaldo coding region and LTR were detected in the A4 clone and a truncated Osvaldo with many substitutions was found in the B9 clone. These altered sequences indicate that the two copies of this retroelement are precolonization insertions. Interestingly, the LTR of the A4 clone and the reverse transcriptase region of B9 show identical sequences in all colonizing populations indicating, most probably, that they are identical by descent. Moreover, Osvaldo is inserted at the same nucleotide site in all colonizing populations. On the other hand an almost identical LTR sequence, except by 1 base deletion, was found in the B4 clone compared to the canonical active Osvaldo element. These results suggest that Osvaldo copies in highly occupied sites are, most probably, identical by descent and strongly favor the founder hypothesis. On the other hand, low-insertion-frequency sites could represent recent transposition events. This work emphasizes the importance of molecular population studies to disentangle the effects of genetic drift and transposition in colonization.
The Overexpression of aSaccharomyces cerevisiaeCentromeric Histone H3 Variant Mutant Protein Leads to a Defect in Kinetochore Biorientation
Authors: Collins Kimberly A , Camahort Raymond , Seidel Chris , Gerton Jennifer L , Biggins Sue
Published: December 2006

Chromosomes segregate using their kinetochores, the specialized protein structures that are assembled on centromeric DNA and mediate attachment to the mitotic spindle. Because centromeric sequences are not conserved, centromere identity is propagated by an epigenetic mechanism. All eukaryotes contain an essential histone H3 variant (CenH3) that localizes exclusively to centromeres. Because CenH3 is required for kinetochore assembly and is likely to be the epigenetic mark that specifies centromere identity, it is critical to elucidate the mechanisms that assemble and maintain CenH3 exclusively at centromeres. To learn more about the functions and regulation of CenH3, we isolated mutants in the budding yeast CenH3 that are lethal when overexpressed. These CenH3 mutants fall into three unique classes: (I) those that localize to euchromatin but do not alter kinetochore function, (II) those that localize to the centromere and disrupt kinetochore function, and (III) those that no longer target to the centromere but still disrupt chromosome segregation. We found that a class III mutant is specifically defective in the ability of sister kinetochores to biorient and attach to microtubules from opposite spindle poles, indicating that CenH3 mutants defective in kinetochore biorientation can be obtained.
Sap1 Promotes the Association of the Replication Fork Protection Complex With Chromatin and Is Involved in the Replication Checkpoint in Schizosaccharomyces pombe
Authors: Noguchi Chiaki , Noguchi Eishi
Published: December 2006

Sap1 is involved in replication fork pausing at rDNA repeats and functions during mating-type switching in Schizosaccharomyces pombe. These two roles are dependent on the ability of Sap1 to bind specific DNA sequences at the rDNA and mating-type loci, respectively. In S. pombe, Swi1 and Swi3 form the replication fork protection complex (FPC) and play important roles in the activation of the replication checkpoint and the stabilization of stalled replication forks. Here we describe the roles of Sap1 in the replication checkpoint. We show that Sap1 is involved in the activation of the replication checkpoint kinase Cds1 and that sap1 mutant cells accumulate spontaneous DNA damage during the S- and G2-phases, which is indicative of fork damage. We also show that sap1 mutants have a defect in the resumption of DNA replication after fork arrest. Sap1 is localized at the replication origin ori2004 and this localization is required for the association of the FPC with chromatin. We propose that Sap1 is required to recruit the FPC to chromatin, thereby contributing to the activation of the replication checkpoint and the stabilization of replication forks.
Xlf1 Is Required for DNA Repair by Nonhomologous End Joining in Schizosaccharomyces pombe
Authors: Cavero Santiago , Chahwan Charly , Russell Paul
Published: December 2006

The accurate repair of DNA double-strand breaks is essential for cell survival and maintenance of genome integrity. Here we describe xlf1+, a gene in the fission yeast Schizosaccharomyces pombe that is required for repair of double-strand breaks by nonhomologous end joining during G1 phase of the cell cycle. Xlf1 is the ortholog of budding yeast Nej1 and human XLF/Cernunnos proteins.
Exploratory Activity in Drosophila Requires thekurtzNonvisual Arrestin
Authors: Liu Lingzhi , Davis Ronald L , Roman Gregg
Published: December 2006

When Drosophila adults are placed into an open field arena, they initially exhibit an elevated level of activity followed by a reduced stable level of spontaneous activity. We have found that the initial elevated component arises from the fly's interaction with the novel arena since: (1) the increased activity is independent of handling prior to placement within the arena, (2) the fly's elevated activity is proportional to the size of the arena, and (3) the decay in activity to spontaneous levels requires both visual and olfactory input. These data indicate that active exploration is the major component of elevated initial activity. There is a specific requirement for the kurtz nonvisual arrestin in the nervous system for both the exploration stimulated by the novel arena and the mechanically stimulated activity. kurtz is not required for spontaneous activity; kurtz mutants display normal levels of spontaneous activity and average the same velocities as wild-type controls. Inhibition of dopamine signaling has no effect on the elevated initial activity phase in either wild-type or krz1 mutants. Therefore, the exploratory phase of open field activity requires kurtz in the nervous system, but is independent of dopamine's stimulation of activity.
Methanol and Egg Yolk as Cryoprotectants for Atlantic Salmon Spermatozoa
Authors: Jodun Wade A , King Kim , Farrell Pat , Wayman William
Published: December 2006

The effects of four extenders on the fertilization rates of eggs fertilized with cryopreserved sperm of Atlantic salmon Salmo salar were tested. We used (1) glucose extender (54.04 g glucose/L and 1.7 g KCl/L) with 5% DMSO, (2) glucose extender with 5% DMSO supplemented with 13.3% egg yolk, (3) glucose extender with 10% methanol, and (4) glucose extender with 10% methanol supplemented with 13.3% egg yolk. Fertilization rates, expressed as the percentage of eyed embryos, ranged from 52.7% to 83.5%. Sperm cryopreserved with the glucose extender and 10% methanol supplemented with 13.3% egg yolk yielded significantly higher fertilization rates (83.5%) than did sperm cryopreserved with the other three extenders. Our fertilization rates compare favorably with those observed for eggs from the same year-class fertilized with fresh milt (81.4%) and reared at the White River National Fish Hatchery. The presence of egg yolk in extenders incorporating 10% methanol provided additional protection to salmonid sperm during the freezing and thawing processes and resulted in an increase in survival from 72.9% to 83.5%. However, the cryoprotective effect of egg yolk may be specific to the individual formulation of extenders. In our trials, glucose and 5% DMSO without egg yolk yielded a 66.9% fertilization rate, while glucose and 5% DMSO supplemented with 13.3% egg yolk produced only 52.7% fertilization after cryopreservation.
Detection of the Mitochondrial Apoptosis‐Induced Channel (MAC) and Its Regulation by Bcl‐2 Family Proteins
Authors: Kinnally Kathleen W. , Martinez‐Caballero Sonia , Dejean Laurent M.
Published: December 2006

Apoptosis is a phenomenon fundamental to higher eukaryotes that is integral to such diverse cellular processes as tissue homeostasis, organogenesis, and response to toxins. The release from mitochondria of apoptotic factors such as cytochrome c is a key step during apoptosis of most cells. Cytochrome c release occurs through the MAC (mitochondrial apoptosis‐induced channel), a pore which forms in the mitochondrial outer membrane during early apoptosis and is exquisitely regulated by the Bcl‐2 family of proteins. This unit presents basic and advanced tools for detecting MAC and defining its regulation by Bcl‐2 family proteins and pharmacological agents. Protocols include the use of time‐lapse video‐microscopy to monitor the onset of apoptosis in living cells and patch‐clamp techniques for mitochondria or proteoliposomes containing mitochondrial proteins, which allow direct detection of MAC. These approaches enable an evaluation of the role of MAC and mitochondria in apoptosis of a variety of cell types by many inducers.
Analysis of Ultrasonic Vocalizations Emitted by Infant Rodents
Authors: Branchi Igor , Santucci Daniela , Alleva Enrico
Published: December 2006

Altricial rodent pups emit ultrasonic vocalizations (USVs), which are whistle‐like sounds with frequencies between 30 and 90 kHz. These signals play an important communicative role in mother‐offspring interaction because they elicit in the dam a prompt response as concerning care‐giving behaviors. To investigate neurobehavioral development, the analysis of the number of USVs presents several advantages: (1) USVs are one of the few responses produced by very young rodents that can be quantitatively analyzed and elicited by quantifiable stimuli; (2) USV emission follows a clear ontogenetic profile from birth to the second to third week of life, thus allowing longitudinal analysis during very early post‐natal ontogeny. The reported role played by several receptor agonists and antagonists in modulating the USV rate makes this measure highly informative in investigating the effects of toxicants and, more generally, psychoactive compounds on the development of selected brain systems.
Identifying Small‐Molecule Modulators of Protein‐Protein Interactions
Authors: Horswill Alexander R. , Benkovic Stephen J.
Published: December 2006

This unit outlines methods for identifying cyclic peptides that inhibit protein‐protein interactions. Proteins of interest are cloned into a two‐hybrid system engineered to operate in reverse, allowing the disruption of a protein complex to be coupled to cell growth. Cyclic peptide libraries are generated using an intein‐based plasmid construct, and the cyclized sequence is randomized using a PCR procedure. By transforming plasmid libraries into host cells containing the two‐hybrid fusions, cyclic peptide inhibitors can be identified by growing the cells under the appropriate selective conditions. A detailed procedure for performing the genetic selection and identifying false positives is provided. Methods for building the two‐hybrid protein fusions and optimizing media conditions, as well as an additional protocol for constructing cyclic peptide libraries are also provided.
Benzodiazepine Binding to GABAAReceptors
Authors: Benke Dietmar , Möhler Hanns
Published: December 2006

This unit describes ligand binding assays for the characterization of the benzodiazepine (BZ) site of GABAAreceptors in the central nervous system. The Basic Protocol provides a procedure for analyzing benzodiazepine binding to GABAAreceptors from brain. The experimental design of the method is suitable for all commercially available radioligands that interact with the BZ site of central GABAAreceptors. By using the appropriate radioligand in combination with particular displacer compounds, individual (or groups of) GABAAreceptor subtypes can be identified. Preparation of membrane suspensions for this procedure is described in a Support Protocol, and five Alternate Protocols are provided for the identification of diazepam‐sensitive GABAAreceptors, the α1‐subtype of diazepam‐sensitive receptors, the α5 subtype of diazepam‐sensitive receptors, both diazepam‐sensitive and diazepam‐insensitive receptors, and diazepam‐insensitive receptors exclusively. Each protocol contains a brief description of the requirements for radioligand binding to these five types of BZ sites.
Overview of Drug Discovery and Development
Authors: Ator Mark A. , Mallamo John P. , Williams Michael
Published: December 2006

This overview unit describes the core activities involved in the drug discovery and development process, from target identification ‐ including preclinical biology, medicinal and process chemistry ‐ to pharmacokinetics and metabolism (ADME), and also activities related to the to the drug approval process. The latter include the activities related to the filing of an IND (Investigational New Drug) application and also Phases I – III of clinical trials that form the basis of an NDA (New Drug Application) submission, as well as post‐marketing Phase IV activities as required by the U.S. Food and Drug Administration (FDA) and the European and Japanese counterparts
One‐Dimensional SDS Gel Electrophoresis of Proteins
Authors: Gallagher Sean R.
Published: December 2006

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, that is, in the presence of sodium dodecyl sulfate (SDS). Both full‐size and minigel formats are detailed. Several modifications are provided for specific applications. For separation of peptides and small proteins, the standard buffers are replaced with either a Tris‐tricine buffer system or a modified Tris buffer in the absence of urea. Continuous SDS‐PAGE is a simplified method in which the same buffer is used for both the gel and electrode solutions and the stacking gel is omitted. Other protocols cover the preparation and use of ultrathin gels and gradient gels, and the simultaneous preparation of multiple gels.
Infection of CD4+ Primary T Cells and Cell Lines, Generation of Chronically Infected Cell Lines, and Induction of HIV Expression
Authors: Vicenzi Elisa , Poli Guido
Published: December 2006

Acute infection of most primary cells and cell lines with HIV depends upon the sequential engagement of CD4 (primary receptor) and a chemokine coreceptor (usually CCR5 or CXCR4) by gp120 Env. Chronically infected cell lines and clones are currently used as sources of virus for infecting other cell types, as “factories” for large‐scale production of virions or viral components, and as model systems for studies of regulation of virus expression. This unit provides detailed protocols for acute in vitro HIV infection of primary T cell blasts, interleukin‐2‐stimulated PBMC, and resting PBMC. The unit also contains information on how to determine the chemokine coreceptor usage of the virus for experimental infections. The use of cell lines as targets of acute infection is also described. Finally, protocols for generating and studying chronically HIV‐infected cell lines are provided.
Calcium Chloride
Authors: Kemp Robert , Keegan Suzanne E.
Published: December 2006

The article contains sections titled:

1.

Introduction

2.

Physical Properties

3.

Chemical Properties

4.

Production

5.

Environmental Protection

6.

Quality Specifications

7.

Chemical Analysis

8.

Storage and Transportation

9.

Uses

10.

Economic Aspects

11.

Toxicology and Occupational Health
Carbides
Authors: Tulhoff Helmut
Published: December 2006

The article contains sections titled:

1.Survey1.1.Saltlike Carbides1.2.Metal‐like Carbides1.3.Diamond‐like Carbides1.4.Carbides of Nonmetallic Elements1.5.Crystal Structure1.6.General Production Processes1.7.Uses2.Metal‐like Carbides of Industrial Importance2.1.Tungsten Carbide2.2.Titanium Carbide2.3.Tantalum Carbide2.4.Niobium Carbide2.5.Zirconium Carbide2.6.Hafnium Carbide2.7.Vanadium Carbide2.8.Chromium Carbide2.9.Molybdenum Carbide3.Mixed Carbides3.1.Tungsten ‐ Titanium Carbide3.2.Other Mixed Carbides3.3.Carbonitrides3.4.Mixed Carbonitrides4.Carbides of the Iron Group and Manganese5.Complex Carbides
Carboxylic Acids, Aliphatic
Authors: Riemenschneider Wilhelm
Published: December 2006

The article contains sections titled:1.Introduction2.Physical Properties3.Chemical Properties4.Natural Sources5.Production5.1.Saturated Monocarboxylic Acids5.1.1.Aldehyde Oxidation5.1.2.Carboxylation of Olefins, Koch Process5.1.3.Oxidation of Alkanes5.1.4.Alkali Fusion of Alcohols5.2.Unsaturated Monocarboxylic Acids6.Environmental Protection7.Quality Specifications and Analysis8.Storage and Transportation9.Uses10.Specific Aliphatic Carboxylic Acids10.1.Butyric Acids10.2.Valeric Acids10.3.Octanoic Acids10.4.2‐Ethylhexanoic Acid10.5.Nonanoic Acids10.6.Isodecanoic Acid10.7.Pivalic Acid10.8.Versatic Acids and Neo Acids10.9.Propiolic Acid11.Trade Names, Economic Aspects12.Toxicology and Occupational Health13.Derivatives13.1.Acyl Halides13.2.Anhydrides13.3.Lactams13.4.Halogenated Carboxylic Acids
Chlorophenoxyalkanoic Acids
Authors: Leng Marguerite L.
Published: December 2006

The article contains sections titled:1.Introduction2.Production3.Quality Specifications4.Uses5.Toxicology and Occupational Health6.Biochemical and Environmental Aspects
Chromium Compounds
Authors: Anger Gerd , Halstenberg Jost , Hochgeschwender Klaus , Scherhag Christoph , Korallus Ulrich , Knopf Herbert , Schmidt Peter , Ohlinger Manfred
Published: December 2006

The article contains sections titled:1.Introduction2.Chromium Ores2.1.Ore Deposits2.2.Ore Beneficiation3.Production of Sodium Dichromate3.1.Alkaline Roasting3.2.Leaching of the Roast3.3.Acidification3.4.Crystallization4.Chromium Oxides4.1.Chromium(III) Oxide and Chromium Hydroxide4.2.Chromium(IV) Oxide (Chromium Dioxide)4.3.Chromium(VI) Oxide5.Chromium(III) Salts5.1.General Properties5.2.Chromium(III) Sulfates and Chrome Tanning Agents5.3.Other Chromium(III) Salts6.Chromic Acids and Chromates(VI)6.1.Chromic Acids6.2.Alkali Chromates and Dichromates6.3.Other Chromates7.Other Chromium Compounds8.Analysis9.Transportation, Storage, and Handling10.Environmental Protection11.Ecotoxicology12.Nutrition13.Toxicology and Occupational Health14.Economic Aspects
Coal Pyrolysis
Authors: Scaroni Alan W. , Khan M. Rashid , Eser Semih , Radovic Ljubisa R.
Published: December 2006

The article contains sections titled:

1.Introduction2.Thermoplastic Properties of Coal2.1.Experimental Techniques2.2.Experimental Variables2.2.1.Coal Rank and Petrographic Composition2.2.2.Heating Rate2.2.3.Particle Size2.2.4.Pressure2.2.5.Gaseous Atmosphere2.2.6.Preoxidation2.2.7.Additives2.3.Development of Thermoplasticity2.3.1.Softening2.3.2.Swelling2.3.3.Resolidification2.4.Coke Microstructure and Mesophase Formation2.4.1.Microscopic Characterization2.4.2.Carbonaceous Mesophase2.4.3.Modification of Optical Texture and Properties3.Yield and Distribution of Pyrolysis Products3.1.Experimental Methods3.2.Experimental Variables3.2.1.Time and Temperature3.2.2.Heating Rate3.2.3.Pressure3.2.4.Particle Size3.2.5.Coal Rank3.3.Mechanism4.Kinetics5.Hydropyrolysis6.Pyrolysis Processes6.1.Low‐Temperature Pyrolysis Processes6.1.1.Early Processes6.1.2.Contemporary Processes6.2.High‐Temperature Processes (Coke‐Making)6.2.1.Coke Ovens6.2.2.Coke Properties6.2.3.Byproducts6.2.4.Formed‐Coke Processes6.2.5.Environmental Aspects6.3.Contemporary Hydropyrolysis and Pyrolysis Processes
Combinatorial Chemistry
Authors: Tiebes Dominika , Peters Stefan
Published: December 2006

The article contains sections titled:

1.Introduction2.Concept of Combinatorial Chemistry3.Methods and Techniques of Combinatorial Synthesis3.1.Synthetic Strategies Towards Combinatorial Libraries3.1.1.Split ‐ Pool Synthesis Towards Combinatorial Libraries3.1.1.1.Techniques using Resin Beads3.1.1.2.Tea‐Bag Method3.1.2.Parallel Synthesis Towards Combinatorial Libraries3.1.2.1.Reaction Apparatus using Resin Beads3.1.2.2.Multipin Technique3.1.2.3.Spatially Addressable Parallel Synthesis on Silica Wafer3.1.3.Reagent Mixture Synthesis Towards Combinatorial Libraries3.2.Synthetic Methodology for Organic Library Construction3.2.1.Solid‐Phase Organic Synthesis3.2.1.1.Solid Support, Linker, and Cleavage Strategies3.2.1.2.Reaction Portfolio3.2.2.Synthesis in Solution and Liquid‐Phase Synthesis4.Characterization of Combinatorial Libraries4.1.Analytical Characterization4.2.Hit Identification in Combinatorial Libraries by High‐Throughput Screening4.2.1.Strategies for Libraries of Compound Mixtures4.2.1.1.On‐Bead Screening4.2.1.2.Deconvolution4.2.1.2.1.Iterative Deconvolution4.2.1.2.2.Deconvolution by Positional Scanning4.2.1.2.3.Deconvolution by Orthogonal Libraries4.2.1.3.Encoding4.2.1.4.Multiple Cleavable Linkers4.2.2.Strategies for Libraries of Separate Single Compounds4.2.3.Conclusion5.Automation and Data Processing5.1.Synthesis Automation and Data Processing5.2.Automated Purification6.Library Design and Diversity Assessment6.1.Diversity Assessment for Selection of Building Blocks or Compound6.2.Iterative Optimization Methods7.Economic Aspects and Industrial Case Studies8.Further Developments8.1.Combinatorial Chemistry and Catalysis8.2.Combinatorial Chemistry and Material Science