bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular mapping of tyrosine-phosphorylated proteins in focal adhesions using fluorescence resonance energy transfer
Authors: Ballestrem Christoph , Erez Noam , Kirchner Joachim , Kam Zvi , Bershadsky Alexander , Geiger Benjamin
Published: February 2006

Microscopy-based fluorescence resonance energy transfer (FRET) provides an opportunity to monitor molecular processes in the natural environment in live cells. Here we studied molecular interactions and tyrosine phosphorylation of paxillin, Crk-associated substrate (CAS), and focal adhesion kinase (FAK) in focal adhesions. For that purpose, these focal adhesion phosphoproteins, fused to cyan or yellow fluorescent proteins (CFP or YFP) were expressed in cultured fibroblasts. To assess the dynamics of tyrosine phosphorylation we used YFP- or CFP-tagged SH2 domain of pp60src (dSH2), which specifically binds to phosphotyrosine residues. FRET measurements, combined with immunolabeling with phosphospecific antibodies revealed that FAK, CAS and paxillin are tyrosine phosphorylated in early matrix adhesions and that FAK is in FRET proximity to CAS and paxillin in focal complexes and focal adhesions. Data suggest that paxillin incorporation into nascent focal complexes precedes its tyrosine phosphorylation, which then gradually increases. In cells treated with Rho-kinase inhibitors or expressing constitutively active Rac, focal complexes showed similar levels of paxillin tyrosine phosphorylation as seen in mature focal adhesions. Dynamic FRET-based examination indicated that paxillin phosphorylation occurs in specific areas (hotspots) within focal adhesions, whereas FAK phosphorylation is broadly distributed.
Ca2+ stimulation of adenylyl cyclase generates dynamic oscillations in cyclic AMP
Authors: Willoughby Debbie , Cooper Dermot M. F.
Published: February 2006

The spatial and temporal complexity of Ca2+ signalling is central to the regulation of a diverse range of cellular processes. The decoding of dynamic Ca2+ signals is, in part, mediated by the ability of Ca2+ to regulate other second messengers, including cyclic AMP (cAMP). A number of kinetic models (including our own) predict that interdependent Ca2+ and cAMP oscillations can be generated. A previous study in Xenopus neurons illustrated prolonged, low-frequency cAMP oscillations during bursts of Ca2+ transients. However, the detection of more dynamic Ca2+ driven changes in cAMP has, until recently, been limited by the availability of suitable cAMP probes with high temporal resolution. We have used a newly developed FRET-based cAMP indicator comprised of the cAMP binding domain of Epac-1 to examine interplay between Ca2+ and cAMP dynamics. This probe was recently used in excitable cells to reveal an inverse relationship between cAMP and Ca2+ oscillations as a consequence of Ca2+-dependent activation of phosphodiesterase 1 (PDE1). Here, we have used human embryonic kidney (HEK293) cells expressing the type 8 adenylyl cyclase (AC8) to examine whether dynamic Ca2+ changes can mediate phasic cAMP oscillations as a consequence of Ca2+-stimulated AC activity. During artificial or agonist-induced Ca2+ oscillations we detected fast, periodic changes in cAMP that depended upon Ca2+ stimulation of AC8 with subsequent PKA-mediated phosphodiesterase 4 (PDE4) activity. Carbachol (10 μM) evoked cAMP transients with a peak frequency of ∼3 minute-1, demonstrating phasic oscillations in cAMP and Ca2+ in response to physiological stimuli. Furthermore, by imposing a range of Ca2+-oscillation frequencies, we demonstrate that AC8 acts as a low-pass filter for high-frequency Ca2+ events, enhancing the regulatory options available to this signalling pathway.
The polarity-establishment component Bem1p interacts with the exocyst complex through the Sec15p subunit
Authors: France Y. Ellen , Boyd Charles , Coleman Jeff , Novick Peter J.
Published: February 2006

Spatial regulation of the secretory machinery is essential for the formation of a new bud in Saccharomyces cerevisiae. Yet, the mechanisms underlying cross-talk between the secretory and the cell-polarity-establishment machineries have not been fully elucidated. Here, we report that Sec15p, a subunit of the exocyst complex, might provide one line of communication. Not only is Sec15p an effector of the rab protein Sec4p, the master regulator of post-Golgi trafficking, but it also interacts with components of the polarity-establishment machinery. We have demonstrated a direct physical interaction between Sec15p and Bem1p, a protein involved in the Cdc42p-mediated polarity-establishment pathway, confirming a prior two-hybrid study. When this interaction is compromised, as in the case of cells lacking the N-terminal 138 residues of Bem1p, including the first Src-homology 3 (SH3) domain, the localization of green fluorescent protein (GFP)-tagged Sec15 is affected, especially in the early stage of bud growth. In addition, Sec15-1p, which is defective in Bem1p binding, mislocalizes along with Sec8p, another exocyst subunit. Overall, our evidence suggests that the interaction of Sec15p with Bem1p is important for Sec15p localization at the early stage of bud growth and, through this interaction, Sec15p might play a crucial role in integrating the signals between Sec4p and the components of the early-polarity-establishment machinery. This, in turn, helps to coordinate the secretory pathway and polarized bud growth.
Seasonal influence of wave action on thread production in Mytilus edulis
Authors: Moeser Gretchen M. , Leba Heather , Carrington Emily
Published: February 2006

The blue mussel Mytilus edulis maintains a strong attachment to the substrate in high energy environments by producing byssal threads. On the shores of Rhode Island, USA, mussel attachment strength increases twofold in spring compared to that in the fall. While many factors could influence attachment strength (temperature, food supply, predator cues, etc.), it has been proposed that the variation observed is primarily due to increased thread production during winter and spring in response to increased wave action. This study evaluates the influence of three aspects of wave action on the thread production of M. edulis. Mussels were exposed to flow, acceleration and byssal loading stimuli and the subsequent number of byssal threads produced in the laboratory was monitored. Increased flow elicited the strongest response, significantly decreasing thread production in mussels. This result was confirmed in flume experiments exposing mussels to a range of flows, with reduced thread production above 15 cm s–1. The influence of both acceleration and byssal loading was sporadic and inconsistent across seasons. Surprisingly, overall thread production in the laboratory was lowest in winter, a time when mussels typically peak in attachment. A similar seasonal pattern was observed in field assays, with high thread production during periods of elevated temperature, reduced wave action,and high reproductive condition. These results suggest that seasonal variation in attachment strength does not reflect increased thread production in response to wave action, and that other possible factors, such as seasonal variability in both the material properties of byssal threads and thread decay rates, warrant further investigation.
Nocturnal colour vision – not as rare as we might think
Authors: Kelber Almut , Roth Lina S. V.
Published: February 2006

The dual retina of humans and most vertebrates consists of multiple types of cone for colour vision in bright light and one single type of rod, leaving these animals colour-blind at night. Instead of comparing the signals from different spectral types of photoreceptors, they use one highly sensitive receptor, thus improving the signal-to-noise ratio. However, nocturnal moths and geckos can discriminate colours at extremely dim light intensities when humans are colour-blind, by sacrificing spatial and temporal rather than spectral resolution. The advantages of colour vision are just as obvious at night as they are during the day. Colour vision is much more reliable than achromatic contrast, not only under changing light intensities, but also under the colour changes occurring during dusk and dawn. It can be expected that nocturnal animals other than moths and geckos make use of the highly reliable colour signals in dim light.
Genetic Analyses Demonstrate That Bone Morphogenetic Protein Signaling Is Required for Embryonic Cerebellar Development
Authors: Qin Lihua , Wine-Lee Lara , Ahn Kyung J. , Crenshaw E. Bryan
Published: February 2006

The cerebellum has been a useful model for studying many aspects of neural development because of its relatively simple cytoarchitecture and developmental program. Yet, the genetic mechanisms underlying early differentiation and patterning of the cerebellum are still poorly characterized. Cell expression studies and culture experiments have suggested the importance of bone morphogenetic proteins (BMPs) in development of specific populations of cerebellar neurons. Here, we examined mice with targeted mutations in the BMP type I receptor genesBmpr1aandBmpr1b, to genetically test the hypothesis that BMPs play an inductive role in the embryogenesis of cerebellar granule cells. InBmpr1a;Bmpr1bdouble knock-out mice, severe cerebellar patterning defects are observed resulting in smaller cerebella that are devoid of foliation. In mutants containing either single BMP receptor gene mutation alone, cerebellar histogenesis appears normal, thereby demonstrating functional redundancy of type I BMP receptors during cerebellar development. Loss of BMP signaling in double mutant animals leads to a dramatic reduction in the number of cerebellar granule cells and ectopic location of many of those that remain. Molecular markers of granule cell specification, includingMath1andZic1, are drastically downregulated. In addition, Purkinje cells are disorganized and ectopically located, but they appear to be correctly specified. Consistent with the interpretation that granule cells alone are affected, phosphorylated Smad1/5/8 is immunolocalized predominantly to granule cell precursors and not appreciably detected in Purkinje cell precursors. This study demonstrates that BMP signaling plays a crucial role in the specification of granule cells during cerebellar development.
The Subfornical Organ: A Central Target for Circulating Feeding Signals
Authors: Pulman Katherine J. , Fry W. Mark , Cottrell G. Trevor , Ferguson Alastair V.
Published: February 2006

The mechanisms through which circulating ghrelin relays hunger signals to the CNS are not yet fully understood. In this study, we have examined the potential role of the subfornical organ (SFO), a circumventricular structure that lacks the normal blood–brain barrier, as a CNS site in which ghrelin acts to influence the hypothalamic centers controlling food intake. We report that ghrelin increased intracellular calcium concentrations in 28% (12 of 43) of dissociated SFO neurons and that the SFO expresses mRNA for the growth hormone secretagogue receptor. Whole-cell patch recordings from SFO neurons demonstrated that in 29% (9 of 31) of neurons tested ghrelin induced a mean depolarization of 7.4 ± 0.69 mV, accompanied by an increase in action potential frequency. Voltage-clamp recordings revealed that ghrelin activates a putative nonselective cationic conductance.

Previous reports that the satiety signal amylin exerts similar excitatory effects on SFO neurons led us to examine whether these two peptides influence different subpopulations of SFO neurons. Concentration-dependent depolarizing effects of amylin were observed in 59% (28 of 47) of SFO neurons (mean depolarization, 8.32 ± 0.60 mV). In contrast to ghrelin, voltage-clamp recordings suggest that amylin influences a voltage-dependent current activated at depolarized potentials. We tested single SFO neurons with both peptides and identified cells responsive only to ghrelin (n= 9) and only to amylin (n= 7) but no cells that responded to both peptides. These data support a role for the SFO as a center at which ghrelin and amylin may influence separate subpopulations of neurons to influence the hypothalamic regulation of feeding.
Glutamate Receptor Exocytosis and Spine Enlargement during Chemically Induced Long-Term Potentiation
Authors: Kopec Charles D. , Li Bo , Wei Wei , Boehm Jannic , Malinow Roberto
Published: February 2006

The changes in synaptic morphology and receptor content that underlie neural plasticity are poorly understood. Here, we use a pH-sensitive green fluorescent protein to tag recombinant glutamate receptors and monitor their dynamics onto dendritic spine surfaces. We show that chemically induced long-term potentiation (chemLTP) drives robust exocytosis of AMPA receptors. In contrast, the same stimulus produces a small reduction of NMDA receptors from the spine surface. chemLTP produces similar modification of small and large spines. Interestingly, during chemLTP induction, spines increase in volume before accumulation of AMPA receptors on their surface, indicating that distinct mechanisms underlie changes in morphology and receptor content.
Physical and Functional Interaction between Protocadherin 15 and Myosin VIIa in Mechanosensory Hair Cells
Authors: Senften Mathias , Schwander Martin , Kazmierczak Piotr , Lillo Concepcion , Shin Jung-Bum , Hasson Tama , Géléoc Gwenaëlle S. G. , Gillespie Peter G. , Williams David , Holt Jeffrey R. , Müller Ulrich
Published: February 2006

Hair cells of the mammalian inner ear are the mechanoreceptors that convert sound-induced vibrations into electrical signals. The molecular mechanisms that regulate the development and function of the mechanically sensitive organelle of hair cells, the hair bundle, are poorly defined. We link here two gene products that have been associated with deafness and hair bundle defects, protocadherin 15 (PCDH15) and myosin VIIa (MYO7A), into a common pathway. We show that PCDH15 binds to MYO7A and that both proteins are expressed in an overlapping pattern in hair bundles. PCDH15 localization is perturbed in MYO7A-deficient mice, whereas MYO7A localization is perturbed in PCDH15-deficient mice. Like MYO7A, PCDH15 is critical for the development of hair bundles in cochlear and vestibular hair cells, controlling hair bundle morphogenesis and polarity. Cochlear and vestibular hair cells from PCDH15-deficient mice also show defects in mechanotransduction. Together, our findings suggest that PCDH15 and MYO7A cooperate to regulate the development and function of the mechanically sensitive hair bundle.
Essential Role of the Main Olfactory System in Social Recognition of Major Histocompatibility Complex Peptide Ligands
Authors: Spehr Marc , Kelliher Kevin R. , Li Xiao-Hong , Boehm Thomas , Leinders-Zufall Trese , Zufall Frank
Published: February 2006

Genes of the major histocompatibility complex (MHC), which play a critical role in immune recognition, influence mating preference and other social behaviors in fish, mice, and humans via chemical signals. The cellular and molecular mechanisms by which this occurs and the nature of these chemosignals remain unclear. In contrast to the widely held view that olfactory sensory neurons (OSNs) in the main olfactory epithelium (MOE) are stimulated by volatile chemosignals only, we show here that nonvolatile immune system molecules function as olfactory cues in the mammalian MOE. Using mice with targeted deletions in selected signal transduction genes (CNGA2,CNGA4), we used a combination of dye tracing, electrophysiological, Ca2+imaging, and behavioral approaches to demonstrate that nonvolatile MHC class I peptides activate subsets of OSNs at subnanomolar concentrationsin vitroand affect social preference of male micein vivo. Both effects depend on the cyclic nucleotide-gated (CNG) channel geneCNGA2, the function of which in the nose is unique to the main population of OSNs. Disruption of the modulatoryCNGA4channel subunit reveals a profound defect in adaptation of peptide-evoked potentials in the MOE. Because sensory neurons in the vomeronasal organ (VNO) also respond to MHC peptides but do not expressCNGA2, distinct mechanisms are used by the mammalian main and accessory olfactory systems for the detection of MHC peptide ligands. These results suggest a general role for MHC peptides in chemical communication even in those vertebrates that lack a functional VNO.
The Art of Reframing Political Debates
Authors: Ryan Charlotte , Gamson William A.
Published: February 2006

Activists cannot build political power simply by framing their message in ways that resonate with broader cultural values. To succeed, framing strategies must be integrated with broader movement-building efforts.
Surrenders and Quagmires
Authors: Wagner-Pacifici Robin
Published: February 2006

There used to be accepted rituals for surrendering, understood by everyone. Today, it is not always clear how and when a conflict ends.
Solution Focused Teaching: A Transformative Approach to Teaching Nursing
Authors: McAllister Margaret , Venturato Lorraine , Johnston Amy , Rowe Jennifer , Tower Marion , Moyle Wendy
Published: February 2006

Nurses work in complex health care systems, which today are based not only on medical treatment and cure, but also on enabling individuals and strengthening communities. This paper describes a curriculum approach to teaching nursing in an Australian university which seeks to prepare students for this environment. Underpinned by transformative education principles, and termed Solution Focused Nursing, specific teaching and learning strategies are discussed. A goal is to cultivate critical thinkers and knowledge workers, that is, nurses who are not only able to work skillfully, strategically, and respectfully with clients, but who also demonstrate discernment, optimism, and vision about nursing and health care.
Measurement of the Auditory Brainstem Response (ABR) to Study Auditory Sensitivity in Mice
Authors: Willott James F.
Published: February 2006

The ABR is an electroencephalographic response measured with scalp electrodes. It provides a quick, easy, and reliable method for physiological assessment of auditory sensitivity in mice. A series of brief tone pips or clicks is presented to an anesthetized mouse at a high rate of speed; each click evokes waves of neural activity in the brainstem that are computer‐averaged so they are differentiated from non‐auditory background voltages. The intensity of the clicks is reduced in steps until an ABR can no longer be discerned, thereby defining the ABR threshold, which is closely related to the hearing threshold. Key procedural issues are: (1) accurate calibration of the acoustics (what sounds arrive at the mouse's ear), (2) anesthetization of the mouse, (3) setting up the recording electrodes, (4) the protocol for presenting acoustic stimuli and obtaining thresholds, and (5) interpretation of ABR data.
Overview of Molecular Genetic Diagnosis
Authors: Korf Bruce R.
Published: February 2006

This unit describes the two approaches for molecular genetic diagnosis, direct mutation analysis and linkage studies. Direct mutation analysis is possible if the gene responsible for a disorder has been cloned and techniques exist to detect mutations. Genetic linkage analysis is used when polymorphic markers are available that are closely linked to a disease gene but the gene itself has not been cloned, or when it is difficult or impossible to detect mutations in the gene. For direct mutation analysis, the unit offers a review of the types of mutations that can be detected and the strategies that can be employed for their detection. The unit then describes the types of disorders that are best suited for linkage‐based analysis and offers guidance for interpreting the data.
Yeast as a Model for Human Disease
Authors: Smith Michael G. , Snyder Michael
Published: February 2006

The sequencing of the human genome promised the identification of disease‐causing genes and, subsequently, therapies for those diseases. However, when identifying the genetic basis of a disease, it is not uncommon to discover an abnormal protein whose normal function is unknown. The genetic manipulations required to assign function to genes is often extremely difficult, if not impossible, in human cells. Model organisms have been used to facilitate understanding of gene function because of the ease of genetic manipulations and because many features of eukaryotic physiology have been conserved across phyla. Yeast is a simple eukaryote with a tractable genome, a short generation time, and a large network of researchers who have generated a vast arsenal of research tools. These traits make yeast ideally suited to help reveal the function of genes implicated in human disease.
Detecting Protein–Protein Interactions with CFP‐YFP FRET by Acceptor Photobleaching
Authors: Karpova Tatiana , McNally James G.
Published: February 2006

FRET is a light microscopy method for detecting protein‐protein interactions within intact cells. The FRET protocol presented here is for CFP‐ and YFP‐tagged proteins examined with an argon laser on a scanning confocal microscope. FRET is assayed by one of the most straightforward approaches available, namely, acceptor photobleaching. In this procedure, the YFP‐tagged protein (the FRET “acceptor”) is photobleached at a cellular site of interest, and then the intensity of the CFP‐tagged protein (the FRET “donor”) at that same site is measured. In principle, FRET is detected when the CFP intensity increases after the photobleaching of YFP. This unit describes the appropriate steps to perform this measurement, as well as the necessary controls to ensure that an increase in CFP intensity, if detected, in fact reflects bona fide FRET. Successful application of the protocol will support the conclusion that the CFP‐ and YFP‐tagged proteins directly interact at the site of the photobleaching
Multiplexed Microsphere‐Based Flow Cytometric Immunoassays
Authors: Kellar Kathryn L. , Mahmutovic Aida J. , Bandyopadhyay Kakali
Published: February 2006

Multiplexed microsphere‐based immunoassays can be developed to simultaneously measure multiple analytes in a biologic system by flow cytometric resolution of spectrally distinct microspheres coupled with capture molecules and reporter fluorochromes bound to detection antibodies. A multiplexed sandwich immunoassay is based on an ELISA format that is transferred directly to microspheres to quantitate multiple antigens. These assays require smaller sample volumes, are less expensive, and are as reproducible, reliable, and sensitive as ELISAs. However, potential cross‐reactivities between multiplexed antibodies, antigens, and specimens need to be systematically eliminated during the validation process. Sandwich and competitive immunoassays, which require only one antigen‐specific antibody, can be combined in the same multiplexed array. Antibody‐capture immunoassays are used to detect multiple antibodies from a specimen for diagnostic or surveillance purposes. The protocols for these three multiplexed immunologic assays are accompanied by methods for coupling analytes to microspheres and biotinylation of antibodies with a water‐soluble derivative.
Detection of Gene Fusions in Acute Leukemia Using Bead Microarrays
Authors: Pihan German
Published: February 2006

This unit describes a method for the rapid and simultaneous detection of hybrid mRNAs (hRNA; also known as gene fusions, RNA fusions, or chimeric RNA), resulting from recurrent chromosome translocations or inversions in acute leukemia. The principal components of the assay are a multiplex RT‐PCR, which simultaneously amplifies any of a number of possible hRNA targets, and a liquid bead microarray (LBMA), which is capable of unambiguously identifying the amplified hRNA. The entire procedure from RNA extraction to display of results can be broken into four steps: (1) conjugated‐microsphere and LBMA manufacture and quality control, (2) cDNA synthesis, (3) multiplex PCR, and (4) hybridization of multiplex PCR products to the hRNA‐detecting LBMA and assay read‐out on a flow cytometer. The assay is designed to detect the most common risk‐stratifying translocation in pediatric acute lymphoblastic leukemia, satisfying the demand for inclusion of genetic data in the diagnosis of acute leukemia as predicated by the current WHO classification of hematopoietic and lymphoid neoplasms.
Mathematical modeling of tin‐free chemically‐active antifouling paint behavior
Authors: Yebra Diego Meseguer , Kiil Søren , Dam‐Johansen Kim , Weinell Claus E.
Published: February 2006

Mathematical modeling has been used to characterize and validate the working mechanisms of tin‐free, chemically‐active antifouling (AF) paints. The model‐based analysis of performance data from lab‐scale rotary experiments has shown significant differences between antifouling technologies as regards the biocide leaching and the surface polishing processes. Hence, the modeling framework developed in this work is built so as to describe any generic, chemically‐active AF paint through model parameters, the values of which can be obtained or adjusted from relatively fast measurements. The detailed quantitative information on reacting AF paint systems obtained can be used for accelerated product optimization purposes, thus facilitating the transition to cleaner antifouling technologies using, for example, fast‐degrading natural or synthetic bioactive components. © 2006 American Institute of Chemical Engineers AIChE J, 2006
Guest Encapsulation and Self‐Assembly of a Cavitand‐Based Coordination Capsule
Authors: Haino Takeharu , Kobayashi Mutsumi , Fukazawa Yoshimasa
Published: February 2006

The synthesis and spectroscopic characterization of a cavitand‐based coordination capsule 1⋅4 BF4 of nanometer dimensions is described. Encapsulation studies of large aromatic guests as well as aliphatic guests were performed by using 1H NMR spectroscopy in [D1]chloroform. In addition to the computational analysis of the shape and geometry of the capsule, an experimental approach to estimate the interior size of the cavity is discussed. The cavity provides a highly rigid binding space in which molecules with lengths of approximately 14 Å can be selectively accommodated. The rigid cavity distinguished slight structural differences in the flexible alkyl‐chain guests as well as the rigid aromatic guests. The detailed thermodynamic studies revealed that not only CH–π interactions between the methyl groups on the guest termini and the aromatic cavity walls, but also desolvation of the inner cavity play a key role in the guest encapsulation. The cavity preferentially selected the hydrogen‐bonded heterodimers of a mixture of two or three carboxylic acids 18–20. The chiral capsule encapsulated a chiral guest to show diastereoselection.
A flow cytometric screening test for detergent‐resistant surface antigens in monocytes
Authors: Wolf Zsuzsanna , Orsó Evelyn , Werner Tobias , Boettcher Alfred , Schmitz Gerd
Published: February 2006

Rafts resemble cholesterol‐ and glycosphingolipid‐enriched, liquid‐ordered plasma membrane microdomains, showing resistance to nonionic detergents, and are involved in various cellular processes. In the present study, we have tested surface antigens on resting and lipopolysaccharide (LPS)‐stimulated human peripheral blood monocytes for their detergent resistance (i.e. raft‐association), by flow cytometry. Constitutive (CD14, CD32, CD55), or LPS‐induced (CD81) raft‐association, and detergent solubility (i.e. exclusion of rafts) (CD71) of monocyte antigens in the presence of 0.01% Triton X‐100 are clearly demonstrated. Flow cytometric detergent insolubility is a powerful tool for rapid screening the raft‐association of monocyte antigens in a whole‐blood assay. © 2006 International Society for Analytical Cytology
Divergent effects of IL‐12 and IL‐23 on the production of IL‐17 by human T cells
Authors: Hoeve Marieke A. , Savage Nigel D. L. , de Boer Tjitske , Langenberg Dennis M. L. , de Waal Malefyt René , Ottenhoff Tom H. M. , Verreck Frank A. W.
Published: February 2006

IL‐23 is regarded as a major pro‐inflammatory mediator in autoimmune disease, a role which until recently was ascribed to its related cytokine IL‐12. IL‐23, an IL‐12p40/p19 heterodimeric protein, binds to IL‐12Rβ1/IL‐23R receptor complexes. Mice deficient for p19, p40 or IL‐12Rβ1 are resistant to experimental autoimmune encephalomyelitis or collagen‐induced arthritis. Paradoxically, however, IL‐12Rβ2‐ and IL‐12p35‐deficient mice show remarkable increases in disease susceptibility, suggesting divergent roles of IL‐23 and IL‐12 in modulating inflammatory processes. IL‐23 induces IL‐17, which mediates inflammation and tissue remodeling, but the role of IL‐12 in this respect remains unidentified. We investigated the roles of exogenous (recombinant) and endogenous (macrophage‐derived) IL‐12 and IL‐23, on IL‐17‐induction in human T‐cells. IL‐23 enhanced IL‐17 secretion, as did IL‐2, IL‐15, IL‐18 and IL‐21. In contrast, IL‐12 mediated specific inhibition of IL‐17 production. These data support the role of IL‐23 in inflammation through stimulating IL‐17 production by T lymphocytes, and importantly indicate a novel regulatory function for IL‐12 by specifically suppressing IL‐17 secretion. These data therefore extend previous reports that had indicated unique functions for IL‐23 and IL‐12 due to distinct receptor expression and signal transduction complexes, and provide novel insights into the regulation of immunity, inflammation and immunopathology.
Critical role for c‐kit (CD117) in T cell lineage commitment and early thymocyte development in vitro
Authors: Massa Steffen , Balciunaite Gina , Ceredig Rhodri , Rolink Antonius G.
Published: February 2006

The precise roles played by the transmembrane receptor tyrosine kinase c‐kit and its ligand stem cell factor in early T cell development are difficult to study. Using cloned Pax5‐deficient progenitor B cells, we show that following Notch signaling, which induces their commitment to the T cell developmental pathway, c‐kit expression is rapidly up‐regulated at both the transcriptional and cell surface level. Using either an anti‐c‐kit monoclonal antibody or Gleevec, a pharmacological inhibitor of c‐kit signaling, we show that the Notch‐induced T cell differentiation of either Pax5‐deficient progenitor B cells, or the equivalent cell from the bone marrow of normal mice, is strictly dependent on c‐kit signaling, whereas the differentiation of normal progenitors into the B cell lineage is not. Moreover, we show that the Notch and IL‐7 signaling‐induced proliferation and differentiation of CD44+CD25–c‐kithigh and CD44+CD25+c‐kithigh thymocytes along the T cell, but not natural killer cell or macrophage, pathway also requires c‐kit signaling, whereas the Notch‐induced proliferation and differentiation of CD44–CD25+c‐kitint cells along the T cell pathway is independent of c‐kit. These results further highlight the complex inter‐relationships existing between c‐kit, Notch and IL‐7 receptor signaling that control the proliferation and differentiation of early T cell progenitors.See accompanying commentary: http://dx.doi.org/10.1002/eji.200635950
Phase Relations Between β‐Tricalcium Phosphate and Hydroxyapatite with Manganese(II): Structural and Spectroscopic Properties
Authors: Mayer Isaac , Cuisinier Frederic J. G. , Popov Ina , Schleich Yechezkel , Gdalya Sarit , Burghaus Olaf , Reinen Dirk
Published: February 2006

The preparation of Mn‐containing β‐tricalcium phosphate (β‐TCP) samples was achieved in two ways: a) transformation of precipitated Mn‐containing calcium hydroxyapatite (HA) to β‐TCP by heating at 1100 °C, and b) preparation by solid‐state reaction of a mixture of CaCO3, (NH4)2HPO4, and Mn(NO3)2 at 1100 °C. Powder X‐ray diffraction (XRD) analyses of the samples, obtained by both methods, show well‐defined patterns with structural data of the rhombohedral R3c, β‐TCP phase. The calculated lattice constants are smaller than those known for β‐Ca3(PO4)2 because of substitution of Ca2+ by Mn2+. EPR spectroscopy indeed reveals that manganese is divalent in the samples. Apparently, the Ca(5) site in the β‐TCP structure is occupied by Mn2+. The distribution of Mn2+ between the β‐TCP and the HA phase in the case of preparation (b) was studied by EPR spectroscopy, and a pronounced preference for the former lattice was found. Micron‐ and submicron‐sized crystals with visible faces were observed by TEM in the case of β‐TCP prepared by solid‐state reaction, and large micron‐sized, droplike‐shaped crystals, sensitive to beam radiation, were found in the case of samples prepared by heating HA at elevated temperatures. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006)
A Synthetic Approach Towards Interlocked π‐Conjugated Macrocycles
Authors: Ammann Martin , Rang Alexander , Schalley Christoph A. , Bäuerle Peter
Published: February 2006

A method for the synthesis of interlocked π‐conjugated macrocycles is described. Starting from 2,9‐bis(oligothienyl)[1,10]phenanthrolines, (trimethylsilyl)acetylene groups were introduced at the terminal thiophene rings by selective iodination and subsequent Sonogashira–Hagihara coupling. Subsequently, we applied our recently developed metal‐template approach to macrocyclization reactions by treating the deprotected acetylenes with cis‐[Pt(dppp)Cl2] to yield a platina‐macrocycle. Based on this synthetic knowledge, by a multiple transition‐metal‐template protocol we accomplished the synthesis of a copper(I)–catenate consisting of two interlocked π‐conjugated macrocycles which contain oligothiophene, diacetylene and phenanthroline units. Conclusive evidence for the new structures came from a detailed characterization of the platina‐macrocycles and catenates by ESI‐FT‐ICR and tandem mass spectrometry experiments. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006)
Numerical Simulation of the Effects of Experimental Error on the Higher Harmonic Components of Fourier Transformed AC Voltammograms
Authors: Gavaghan D. J. , Bond A. M.
Published: February 2006

In this simulation study we investigate the effects of realistic levels of random and systematic experimental error in higher harmonics detected by Fourier transformed AC voltammetry for a reversible process. We show that by analyzing in the frequency domain results that are collected in the time domain, we can achieve a very large reduction in the magnitude of random (Gaussian) noise, and complete elimination of the systematic error introduced by mains frequency in both the small and large amplitude cases. As a consequence, we confirm experimental observations that even the eighth harmonic for a reversible process can be readily detected in the presence of very significant noise levels when 100 mV amplitude sine wave perturbations are employed.
Reference Electrode for Ionic Liquids
Authors: Saheb Amir , Janata Jiří , Josowicz Mira
Published: February 2006

Reference electrodes for room temperature ionic liquid (RTIL) applications were constructed that have a known and reproducible potential versus the ferrocene/ferrocenium couple. They are based on reference electrodes of the first kind, Ag/Ag+ couple type, or of the second kind, based on Ag/AgCl in M+Cl−. The former uses AgNO3 salt and the latter tetrabutylammonium chloride, Bu4NCl, dissolved in acetonitrile which are then introduced to the ionic liquid of choice for a final concentration of 0.1 M. The reference electrodes can be easily and reproducibly constructed. An ionic contact of these reference systems with the test electrolyte was made using an asbestos fiber liquid junction. The internal compartment of the reference system was filled with the same ionic liquid as used for the electrochemical experiment. The performance of these reference electrodes was tested in selected ionic liquids using the ferrocene/ferrocenium redox couple. The stability, reproducibility, and temperature behavior of the two reference systems have been characterized in the following ionic liquids: 1‐butyl‐3‐methylimidazolium tetrafluoroborate (BMIBF4), 1‐butyl‐3‐methylimidazolium bis(trifluoromethanesulfonyl)imide (BMI(CF3SO2)2N), and 1‐butyl‐3‐methylimidazolium hexafluorophosphate (BMIPF6). It has been found that the formal potentials of the examined reference systems are stable over several days. There is a linear relationship for the temperature studied in the range from 25 to 60 °C.
Toll‐like receptor 3 on adult human astrocytes triggers production of neuroprotective mediators
Authors: Bsibsi Malika , Persoon‐Deen Carla , Verwer Ronald W.H. , Meeuwsen Sonja , Ravid Rivka , Van Noort Johannes M.
Published: February 2006

Toll‐like receptors (TLRs) are innate immunity receptors that are expressed on a wide range of cell types, including CNS glial cells. In general, TLR engagement by specific sets of microbial ligands triggers production of pro‐inflammatory factors and enhances antigen‐presenting cell functions. The functional roles of TLR in the CNS, however, are still poorly understood. While adult human astrocytes in culture dominantly express TLR4, they display a strikingly strong and selective induction of TLR3 when activated by pro‐inflammatory cytokines, TLR3 or TLR4 agonists, or oxidative stress. Gene profiling analysis of the astrocyte response to either TLR3 or TLR4 activation revealed that TLR3, but not TLR4, induces expression of a range of neuroprotective mediators and several other molecules that regulate cellular growth, differentiation, and migration. Also, TLR3 triggered enhanced production of anti‐inflammatory cytokines including interleukin‐9 (IL‐9), IL‐10, and IL‐11 and downregulation of the p40 subunit of IL‐12 and IL‐23. The collective TLR3‐induced products were found in functional assays to inhibit astrocyte growth, promote human endothelial cell growth, and importantly, to enhance neuronal survival in organotypic human brain slice cultures. Together, our data indicate that TLR3 is induced on human astrocytes upon inflammation and when activated, mediates a comprehensive neuroprotective response rather than a polarized pro‐inflammatory reaction. © 2006 Wiley‐Liss, Inc.
Tumor–stromal interactions reciprocally modulate gene expression patterns during carcinogenesis and metastasis
Authors: Montel Valérie , Mose Evangeline Sari , Tarin David
Published: February 2006

This study used a unique xenogeneic breast cancer model to study the effects of tumor cells and neighboring host cells upon each other in tumor growth and metastasis. It exploited species differences between the interacting components to determine how the host influenced the tumor and vice versa. It was found that the gene expression profiles of highly and poorly metastatic clones from the same human breast carcinoma changed differentially when the cells were transferred from growth in vitro to the mammary gland. We describe novel sets of genes, validated by human‐specific probes, which were induced in the 2 isogenic, but phenotypically different, tumor lineages by the mammary environment. Conversely, the tumor cells also induced changes in gene expression in the neighboring host stromal (i.e., mesenchymal) cell lineages, validated by mouse‐specific probes. Reciprocal inductive interactions were also demonstrated in the tumor deposits formed preferentially in the lungs and lymph nodes by the highly metastatic tumor cells. Subtraction of the induced gene changes in the primary site from those in the metastases revealed that the number and magnitude of specific gene inductions in colonized organs were moderate. This finding indicates that the gene expression program causing metastasis has only limited flexibility and fits well with clinical observations that tumor cells form metastases preferentially in select organs, although tumor cells are scattered ubiquitously. This dependency on suitable host niches suggests new molecular therapeutic avenues that target genes in the host‐support system that is manipulated by the malignant cells. © 2006 Wiley‐Liss, Inc.
Distribution of Trichoptera communities in the Hozgargantacatchment (Los Alcornocales Natural Park, SW Spain)
Authors: Ruiz García Antonio , Herrera Grao Antonio F. , Ferreras‐Romero Manuel
Published: February 2006

The distribution of Trichoptera of the Hozgarganta River (Los Alcornocales Natural Park, SW Spain) in relation with environmental factors was examined.

Three groups of species were recognised according to the altitudinal gradient. In the headwaters the caddisflies Rhyacophila fonticola, Lepidostoma hirtum, Silonella aurata, Allogamus gibraltaricus, Hydropsyche infernalis and Diplectrona felix predominated; in the constrained section of the tributaries Polycentropus kingi, Chimarra marginata, Hydropsyche iberomaroccana, R. fonticola and Tinodes sp. prevailed; finally, in the main channel H. iberomaroccana, C. marginata, Hydropsyche lobata, Leptocerus lusitanicus and Rhyacophila munda were the most important species. A direct ordination analysis (CCA) was used to describe assemblage changes among sites and corroborated that conductivity and temperature were the variables that best explained Trichoptera distribution.

The temporal analysis showed changes in the Trichoptera diversity and richness in permanent stretches, as well as variations in the structure of the communities according to the season. We identified autumn‐winter species (H. infernalis, H. siltalai, H. lobata, R. fonticola and R. munda ) and summer ones (Ithytrichia sp, Oxyethira unidentata, Mystacides azurea and Setodes argentipunctellus ). In the basin we distinguished permanent, intermittent and ephemeral reaches with similar caddisfly richness and diversity, however the species composition associated with each one was different. (© 2006 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Seasonal Dynamics of Picocyanobacteria and Picoeukaryotes in a Large Shallow Lake (Lake Balaton, Hungary)
Authors: Mózes Andrea , Présing Mátyás , Vörös Lajos
Published: February 2006

The abundance and composition of autotrophic picoplankton (APP) were studied between February 2003 and March 2004 in Lake Balaton. Water samples were taken fortnightly in the eutrophic western basin and mesotrophic eastern basin. Our study, which took more than one year, revealed pronounced seasonal pattern of the picoplankton abundance and composition. According to our results there were three types of picoplankton in Lake Balaton: 1. Phycoerythrin‐rich coccoid cyanobacteria (PE), dominant summer picoplankters in the mesotrophic lake area; 2. Phycocyanin‐rich cyanobacteria (PC), the most abundant summer picoplankters in the eutrophic lake area; 3. Picoeukaryotes, dominant winter picoplankters in the whole lake. The observed abundance of picoeukaryotes (3 × 105 cells ml–1) was one of the highest ever found. Our study confirms that in Lake Balaton the colonial autotrophic picoplankton (colonial APP) become dominant in summer in the nutrient limited period. We have found strong negative relationship between the concentrations of available nitrogen forms (NH4–N, NO3–N, urea‐N) and the colonial APP abundance. (© 2006 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Influence of gel properties on neocartilage formation by auricular chondrocytes photoencapsulated in hyaluronic acid networks
Authors: Chung Cindy , Mesa John , Randolph Mark A. , Yaremchuk Michael , Burdick Jason A.
Published: February 2006

The objective of this study was to determine how changes in the network structure and properties of hyaluronic acid (HA) hydrogels, due to variations in the macromer molecular weight (50–1100 kDa) and macromer concentration (2–20 wt %), affect neocartilage formation by encapsulated auricular chondrocytes. To investigate tissue formation, swine auricular chondrocytes were photoencapsulated in the various networks, implanted subcutaneously in the dorsum of nude mice, and explanted after 6 and 12 weeks for biochemical and histological analysis. After 12 weeks, the various constructs were 81–93% water, contained between 0.1 × 106 and 0.6 × 106 chondrocytes per sample, and consisted of 0–0.049 μg chondroitin sulfate/μg wet weight (glycosaminoglycan (GAG) content) and 0.002–0.060 μg collagen/μg wet weight. Histological staining showed an even distribution of chondrocytes and GAGs in addition to minimal type I collagen staining and intense and uniform type II collagen staining in the constructs with greatest neocartilage production. Hydrogels fabricated from 2 wt % of the 50 kDa HA macromer most resembled the properties of native cartilage and show the greatest promise for continued development for cartilage regeneration. © 2006 Wiley Periodicals, Inc. J Biomed Mater Res, 2006
Restricted b‐factors in bipartite graphs and t‐designs
Authors: Arámbula Ivette , Hicks Illya V.
Published: February 2006

We present a new equivalence result between restricted b‐factors in bipartite graphs and combinatorial t‐designs. This result is useful in the construction of t‐designs by polyhedral methods. We propose a novel linear integer programming formulation, which we call GDP, for the problem of finding t‐designs that has a noteworthy advantage compared to the traditional set‐covering formulation. We analyze some polyhedral properties of GPD, implement a branch‐and‐cut algorithm using it and solve several instances of small designs to compare with another point‐block formulation found in the literature. © 2006 Wiley Periodicals, Inc. J Combin Designs 14: 169–182, 2006
Expression profiles of micro RNA in proliferating and differentiating 32D murine myeloid cells
Authors: Shi Bin , Prisco Marco , Calin George , Liu Chang‐gong , Russo Giuseppe , Giordano Antonio , Baserga Renato
Published: February 2006

32D cells are murine myeloid cells that grow indefinitely in Interleukin‐3 (IL‐3). In these cells, the type 1 insulin‐like growth factor (IGF‐I) and granulocytic‐colony stimulating factor (G‐CSF) induce differentiation to granulocytes. 32D cells do not express insulin receptor substrate‐1 (IRS‐1) or IRS‐2, docking proteins of the IGF‐I receptor. Ectopic expression of IRS‐1 in these cells inhibits differentiation, the cells become IL‐3 independent and IGF‐1 dependent and can form tumors in mice. 32D and 32D‐derived cells offer a good model in which to study the expression profiles of Micro Rna (miR) related to sustained proliferation or differentiation. We present here the data obtained with miR micro‐arrays and identify the miR that are regulated by IGF‐1 or G‐CSF and are associated with either differentiation or indefinite cell proliferation of 32D murine myeloid cells. © 2006 Wiley‐Liss, Inc.
Characterizing medullary and human mesenchymal stem cell‐derived adipocytes
Authors: MacKay Danielle L. , Tesar Paul J. , Liang Li‐Nuo , Haynesworth Stephen E.
Published: February 2006

Throughout postnatal years, medullary adipocytes (MAs) increase in both number and size; however, knowledge of these cells pales in comparison to that of other adipocyte depots. It is widely hypothesized that MAs derive from multipotent progenitor cells of the bone marrow, such as human mesenchymal stem cells (hMSCs). Nevertheless, there is a paucity of comparative, molecular‐level studies in support of this hypothesis. In the present article, RTPCR was used to examine similarities and differences in gene expression among MAs, hMSC‐derived adipocytes, and subcutaneous adipocytes. While little or no message for lineage‐specific markers was detected in undifferentiated hMSCs, the data demonstrate that hMSC‐derived adipocytes, MAs, and subcutaneous adipocytes commonly express mRNA encoding for adipogenic transcription factors (PPARγ2, C/EBPα, and SREBP1), adipokines (adipsin, leptin, APM1, and angiotensinogen), and lipid‐metabolizing agents (aP2 and LPL), among other genes. None of the cell populations examined expressed a detectable level of the brown fat marker UCP1. This suggests highly similar gene expression between human subcutaneous and MAs, not previously substantiated to this degree. Coupled with the hMSC‐derived adipocyte analysis, these data provide a framework ultimately for characterizing MAs and identifying their origin and function. J. Cell. Physiol. © 2006 Wiley‐Liss, Inc.