bluuebunny/crossref_metadata_2025 huggingface dataset:
Abnormal Features in Skeletal Muscle from Mice Lacking Mitsugumin29
Authors: Nishi Miyuki , Komazaki Shinji , Kurebayashi Nagomi , Ogawa Yasuo , Noda Tetsuo , Iino Masamitsu , Takeshima Hiroshi
Published: July 2002

Physiological roles of the members of the synaptophysin family, carrying four transmembrane segments and being basically distributed on intracellular membranes including synaptic vesicles, have not been established yet. Recently, mitsugumin29 (MG29) was identified as a novel member of the synaptophysin family from skeletal muscle. MG29 is expressed in the junctional membrane complex between the cell surface transverse (T) tubule and the sarcoplasmic reticulum (SR), called the triad junction, where the depolarization signal is converted to Ca2+ release from the SR. In this study, we examined biological functions of MG29 by generating knockout mice. The MG29-deficient mice exhibited normal health and reproduction but were slightly reduced in body weight. Ultrastructural abnormalities of the membranes around the triad junction were detected in skeletal muscle from the mutant mice, i.e., swollen T tubules, irregular SR structures, and partial misformation of triad junctions. In the mutant muscle, apparently normal tetanus tension was observed, whereas twitch tension was significantly reduced. Moreover, the mutant muscle showed faster decrease of twitch tension under Ca2+-free conditions. The morphological and functional abnormalities of the mutant muscle seem to be related to each other and indicate that MG29 is essential for both refinement of the membrane structures and effective excitation-contraction coupling in the skeletal muscle triad junction. Our results further imply a role of MG29 as a synaptophysin family member in the accurate formation of junctional complexes between the cell surface and intracellular membranes.
Subcellular Compartmentalization of E2f Family Members Is Required for Maintenance of the Postmitotic State in Terminally Differentiated Muscle
Authors: Gill R. Montgomery , Hamel Paul A.
Published: July 2002

Maintenance of cells in a quiescent state after terminal differentiation occurs through a number of mechanisms that regulate the activity of the E2F family of transcription factors. We report here that changes in the subcellular compartmentalization of the E2F family proteins are required to prevent nuclei in terminally differentiated skeletal muscle from reentering S phase. In terminally differentiated L6 myotubes, E2F-1, E2F-3, and E2F-5 were primarily cytoplasmic, E2F-2 was nuclear, whereas E2F-4 became partitioned between both compartments. In these same cells, pRB family members, pRB, p107, and p130 were also nuclear. This compartmentalization of the E2F-1 and E2F-4 in differentiated muscle cells grown in vitro reflected their observed subcellular location in situ. We determined further that exogenous E2F-1 or E2F-4 expressed in myotubes at levels fourfold greater than endogenous proteins compartmentalized identically to their endogenous counterparts. Only when overexpressed at higher levels was inappropriate subcellular location for these proteins observed. At these levels, induction of the E2F-regulated genes, cyclins A and E, and suppression of factors associated with myogenesis, myogenin, and p21Cip1was observed. Only at these levels of E2F expression did nuclei in these terminally differentiated cells enter S phase. These data demonstrate that regulation of the subcellular compartmentalization of E2F-family members is required to maintain nuclei in a quiescent state in terminally differentiated cells.
Mitotic Transcription Repression in Vivo in the Absence of Nucleosomal Chromatin Condensation
Authors: Spencer Charlotte A. , Kruhlak Michael J. , Jenkins Heather L. , Sun Xuejun , Bazett-Jones David P.
Published: July 2002

All nuclear RNA synthesis is repressed during the mitotic phase of the cell cycle. In addition, RNA polymerase II (RNAP II), nascent RNA and many transcription factors disengage from DNA during mitosis. It has been proposed that mitotic transcription repression and disengagement of factors are due to either mitotic chromatin condensation or biochemical modifications to the transcription machinery. In this study, we investigate the requirement for chromatin condensation in establishing mitotic transcription repression and factor loss, by analyzing transcription and RNAP II localization in mitotic cells infected with herpes simplex virus type 1. We find that virus-infected cells enter mitosis and that mitotic viral DNA is maintained in a nucleosome-free and noncondensed state. Our data show that RNAP II transcription is repressed on cellular genes that are condensed into mitotic chromosomes and on viral genes that remain nucleosome free and noncondensed. Although RNAP II may interact indirectly with viral DNA during mitosis, it remains transcriptionally unengaged. This study demonstrates that mitotic repression of transcription and loss of transcription factors from mitotic DNA can occur independently of nucleosomal chromatin condensation.
The Golgi-Associated Hook3 Protein Is a Member of a Novel Family of Microtubule-Binding Proteins
Authors: Walenta Jason H. , Didier Aaron J. , Liu Xinran , Krämer Helmut
Published: July 2002

Microtubules are central to the spatial organization of diverse membrane-trafficking systems. Here, we report that Hook proteins constitute a novel family of cytosolic coiled coil proteins that bind to organelles and to microtubules. The conserved NH2-terminal domains of Hook proteins mediate attachment to microtubules, whereas the more divergent COOH-terminal domains mediate the binding to organelles. Human Hook3 bound to Golgi membranes in vitro and was enriched in the cis-Golgi in vivo. Unlike other cis-Golgi–associated proteins, however, a large fraction of Hook3 maintained its juxtanuclear localization after Brefeldin A treatment, indicating a Golgi-independent mechanism for Hook3 localization. Because overexpression of Hook3 caused fragmentation of the Golgi complex, we propose that Hook3 participates in defining the architecture and localization of the mammalian Golgi complex.
Magi-1c
Authors: Strochlic Laure , Cartaud Annie , Labas Valérie , Hoch Werner , Rossier Jean , Cartaud Jean
Published: July 2002

The muscle-specific receptor tyrosine kinase (MuSK) forms part of a receptor complex, activated by nerve-derived agrin, that orchestrates the differentiation of the neuromuscular junction (NMJ). The molecular events linking MuSK activation with postsynaptic differentiation are not fully understood. In an attempt to identify partners and/or effectors of MuSK, cross-linking and immunopurification experiments were performed in purified postsynaptic membranes from the Torpedo electrocyte, a model system for the NMJ. Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) analysis was conducted on both cross-link products, and on the major peptide coimmunopurified with MuSK; this analysis identified a polypeptide corresponding to the COOH-terminal fragment of membrane-associated guanylate kinase (MAGUK) with inverted domain organization (MAGI)-1c. A bona fide MAGI-1c (150 kD) was detected by Western blotting in the postsynaptic membrane of Torpedo electrocytes, and in a high molecular mass cross-link product of MuSK. Immunofluorescence experiments showed that MAGI-1c is localized specifically at the adult rat NMJ, but is absent from agrin-induced acetylcholine receptor clusters in myotubes in vitro. In the central nervous system, MAGUKs play a primary role as scaffolding proteins that organize cytoskeletal signaling complexes at excitatory synapses. Our data suggest that a protein from the MAGUK family is involved in the MuSK signaling pathway at the vertebrate NMJ.
A Role for CD4 in Peripheral T Cell Differentiation
Authors: Brown Daniel R. , Moskowitz Naomi H. , Killeen Nigel , Reiner Steven L.
Published: July 2002

Naive CD4+ T helper cells (Th) differentiate into one of two well-defined cell types during immune responses. Mature Th1 and Th2 cells regulate the type of response as a consequence of the unique cytokines that they secrete. CD4 serves a prominent role in potentiating antigen recognition by helper T cells. We have examined the role of CD4 in peripheral T cell differentiation by studying helper T cells from mice with a congenital defect in CD4 expression. After protein immunization or infection with Leishmania major, CD4-deficient mice were incapable of mounting antigen-specific Th2 responses, but retained their Th1 potency. CD4-deficient, T cell receptor transgenic T cells were also incapable of Th2 differentiation after in vitro activation. Expression of a wild-type CD4 transgene corrected the Th2 defect of CD4-deficient mice in all immune responses tested. To investigate the role of the cytoplasmic domain, mice reconstituted with a truncated CD4 molecule were also studied. Expression of the tailless CD4 transgene could not rescue the Th2 defect of CD4-deficient mice immunized with protein or CD4-deficient transgenic T cells activated in vitro, raising the possibility that the cytoplasmic domain of CD4 may influence Th2 generation. Expression of the tailless transgene was, however, capable of restoring Th2 development in CD4-deficient mice infected with L. major or CD4-deficient transgenic T cells activated in the presence of recombinant IL-4, demonstrating that the cytoplasmic domain is not absolutely required for Th2 development. Together, these results demonstrate a previously undescribed role of the CD4 molecule. The requirement for CD4 in Th2 maturation reflects the importance of molecules other than cytokines in the control of helper T cell differentiation.
Identification of Putative Cytoskeletal Protein Homologues in the Protozoan Host Hartmannella vermiformis as Substrates for Induced Tyrosine Phosphatase Activity upon Attachment to the Legionnaires' Disease Bacterium, Legionella pneumophila
Authors: Venkataraman Chandrasekar , Gao Lian-Yong , Bondada Subbarao , Kwaik Yousef Abu
Published: July 2002

The Legionnaires' disease bacterium, Legionella pneumophila, is a facultative intracellular pathogen that invades and replicates within two evolutionarily distant hosts, free living protozoa and mammalian cells. Invasion and intracellular replication within protozoa are thought to be major factors in the transmission of Legionnaires' disease. We have recently reported the identification of a galactose/N-acetyl-d-galactosamine (Gal/GalNAc) lectin in the protozoan host Hartmannella vermiformis as a receptor for attachment and invasion by L. pneumophila (Venkataraman, C., B.J. Haack, S. Bondada, and Y.A. Kwaik. 1997. J. Exp. Med. 186:537–547). In this report, we extended our studies to the effects of bacterial attachment and invasion on the cytoskeletal proteins of H. vermiformis. We first identified the presence of many protozoan cytoskeletal proteins that were putative homologues to their mammalian counterparts, including actin, pp125FAK, paxillin, and vinculin, all of which were basally tyrosine phosphorylated in resting H. vermiformis. In addition to L. pneumophila–induced tyrosine dephosphorylation of the lectin, bacterial attachment and invasion was associated with tyrosine dephosphorylation of paxillin, pp125FAK, and vinculin, whereas actin was minimally affected. Inhibition of bacterial attachment to H. vermiformis by Gal or GalNAc monomers blocked bacteria-induced tyrosine dephosphorylation of detergent-insoluble proteins. In contrast, inhibition of bacterial invasion but not attachment failed to block bacteria-induced tyrosine dephosphorylation of H. vermiformis proteins. This was further supported by the observation that 10 mutants of L. pneumophila that were defective in invasion of H. vermiformis were capable of inducing tyrosine dephosphorylation of H. vermiformis proteins. Entry of L. pneumophila into H. vermiformis was predominantly mediated by noncoated receptor-mediated endocytosis (93%) but coiling phagocytosis was infrequently observed (7%). We conclude that attachment but not invasion by L. pneumophila into H. vermiformis was sufficient and essential to induce protein tyrosine dephosphorylation in H. vermiformis. These manipulations of host cell processes were associated with, or followed by, entry of the bacteria by a noncoated receptor-mediated endocytosis. A model for attachment and entry of L. pneumophila into H. vermiformis is proposed.
Regulation of Interleukin (IL)-12 Receptor β2 Subunit Expression by Endogenous IL-12: A Critical Step in the Differentiation of Pathogenic Autoreactive T Cells
Authors: Chang John T. , Shevach Ethan M. , Segal Benjamin M.
Published: July 2002

The interleukin (IL)-12 receptor (R)β2 subunit is the critical molecule involved in maintaining IL-12 responsiveness and controlling T helper cell type 1 lineage commitment. We demonstrate that IL-12 and interferon (IFN)-γ play separate, but complementary, roles in regulating IL-12Rβ2 expression on antigen-specific CD4+ T cells. These results are consistent with our previous observation that IL-12 can promote autoimmune disease through IFN-γ–independent as well as –dependent pathways. Therefore, we compared the induction of IL-12 by, and the expression of the IL-12Rβ2 subunit on, myelin basic protein (MBP)-specific T cells from experimental allergic encephalomyelitis (EAE)-susceptible SJL (H-2s) mice and from EAE- resistant B10.S mice (H-2s). B10.S mice had an antigen-specific defect in their capacity to upregulate the IL-12Rβ2 subunit. Defective expression was not secondary to the production of suppressive cytokines, but to a failure of B10.S MBP-specific T cells to upregulate CD40 ligand expression and to induce the production of IL-12. IL-12Rβ2 expression as well as encephalitogenicity of these cells could be restored by the addition of IL-12. These results suggest that the development of immunotherapies that target the IL-12Rβ2 subunit may be useful for the treatment of autoimmune diseases.
The Majority of H2-M3 Is Retained Intracellularly in a Peptide-Receptive State and Traffics to the Cell Surface in the Presence of N-Formylated Peptides
Authors: Chiu Nancy M. , Chun Taehoon , Fay Miriam , Mandal Manas , Wang Chyung-Ru
Published: July 2002

We used a new monoclonal antibody (mAb 130) to analyze the intracellular trafficking and surface expression of H2-M3, the major histocompatibility complex class Ib molecule that presents N-formylated peptides to cytotoxic T cells. M3 surface expression is undetectable in most cell types due to the paucity of endogenous antigen. M3 is induced on the cell surface by addition of high-affinity N-formylated peptides from mitochondria and listeria. Peptide-induced M3 expression is most efficient on antigen presenting cells. Basal and inducible expression of M3 is transporter associated with antigen processing (TAP)-dependent, distinguishing M3 from the class Ib molecules TL and CD1. Unlike the expression of class Ia molecules and a previously described M3/Ld chimera, surface expression of M3 cannot be rescued by lowered temperature, suggesting that the α3 domain and transmembrane region of M3 may control trafficking. Pulse–chase analysis and use of trafficking inhibitors revealed a pool of empty M3 in the endoplasmic reticulum or early Golgi apparatus. Addition of exogenous peptide allows maturation with kinetics matching those of Dd. The lack of endogenous N-formylated peptide allows discovery of novel pathogen-derived peptides in normal antigen presenting cells. The nonpolymorphic nature of M3 and its ability to present bacterial antigens rapidly and dominantly make it an attractive target for peptide vaccination strategies.
Joining Chain–Expressing and–Nonexpressing B Cell Populations in the Mouse
Authors: Erlandsson Lena , Akerblad Peter , Vingsbo-Lundberg Carina , Kallberg Eva , Lycke Nils , Leanderson Tomas
Published: July 2002

The diphtheria toxin A chain (DTA) was gene targeted into the Joining chain (J chain) locus to create a mouse strain selecting against J chain–expressing cells, JDTA mice. Serum immunoglobulin (Ig)M and serum IgG were reduced six to eightfold, while serum IgA was elevated 14-fold in these mice. JDTA mice were immune competent although the serum Ig response compared with wild-type mice was reduced sixfold at day 14 but only fourfold at day 45 after immunization. Exchanging the DTA gene with a cDNA for c-myc resulted in mice with a distinct phenotype with increased Ig production and enhanced humoral immune responses. Analysis of single B cells stimulated by lipopolysaccharide in vitro using reverse transcription–polymerase chain reaction showed that J chain–nonexpressing B cells could be detected that had a secretory phenotype as determined by an abundance of transcript for secretory IgM. Finally, limiting dilution analysis of peripheral B cells showed that J chain expression was a clonal property already established in naive, peripheral B lymphocytes.
Characterization of T Cell Differentiation in the Murine Gut
Authors: Lambolez Florence , Azogui Orly , Joret Anne-Marie , Garcia Corinne , von Boehmer Harald , Di Santo James , Ezine Sophie , Rocha Benedita
Published: July 2002

Gut intraepithelial CD8 T lymphocytes (T-IEL) are distinct from thymus-derived cells and are thought to derive locally from cryptopatch (CP) precursors. The intermediate stages of differentiation between CP and mature T-IEL were not identified, and the local differentiation process was not characterized. We identified and characterized six phenotypically distinct lineage-negative populations in the CP and the gut epithelium: (a) we determined the kinetics of their generation from bone marrow precursors; (b) we quantified CD3-ϵ, recombination activating gene (Rag)-1, and pre-Tα mRNAs expression at single cell level; (c) we characterized TCR-β, -γ, and -α locus rearrangements; and (d) we studied the impact of different mutations on the local differentiation. These data allowed us to establish a sequence of T cell precursor differentiation in the gut. We also observed that the gut differentiation varied from that of the thymus by a very low frequency of pre-Tα chain mRNA expression, a different kinetics of Rag-1 mRNA expression, and a much higher impact of CD3 ϵ/δ and pre-Tα deficiencies. Finally, only 3% of CP cells were clearly involved in T cell differentiation, suggesting that these structures may have additional physiological roles in the gut.
Vav1 Transduces T Cell Receptor Signals to the Activation of Phospholipase C-γ1 via Phosphoinositide 3-Kinase-dependent and -independent Pathways
Authors: Reynolds Lucinda F. , Smyth Lesley A. , Norton Trisha , Freshney Norman , Downward Julian , Kioussis Dimitris , Tybulewicz Victor L.J.
Published: July 2002

Vav1 is a signal transducing protein required for T cell receptor (TCR) signals that drive positive and negative selection in the thymus. Furthermore, Vav1-deficient thymocytes show greatly reduced TCR-induced intracellular calcium flux. Using a novel genetic system which allows the study of signaling in highly enriched populations of CD4+CD8+ double positive thymocytes, we have studied the mechanism by which Vav1 regulates TCR-induced calcium flux. We show that in Vav1-deficient double positive thymocytes, phosphorylation, and activation of phospholipase C-γ1 (PLCγ1) is defective. Furthermore, we demonstrate that Vav1 regulates PLCγ1 phosphorylation by at least two distinct pathways. First, in the absence of Vav1 the Tec-family kinases Itk and Tec are no longer activated, most likely as a result of a defect in phosphoinositide 3-kinase (PI3K) activation. Second, Vav1-deficient thymocytes show defective assembly of a signaling complex containing PLCγ1 and the adaptor molecule Src homology 2 domain–containing leukocyte phosphoprotein 76. We show that this latter function is independent of PI3K.
Restrictions Limiting the Generation of DNA Double Strand Breaks during Chromosomal V(D)J Recombination
Authors: Tillman Robert E. , Wooley Andrea L. , Hughes Maureen M. , Wehrly Tara D. , Swat Wojciech , Sleckman Barry P.
Published: July 2002

Antigen receptor loci are composed of numerous variable (V), diversity (D), and joining (J) gene segments, each flanked by recombination signal sequences (RSSs). The V(D)J recombination reaction proceeds through RSS recognition and DNA cleavage steps making it possible for multiple DNA double strand breaks (DSBs) to be introduced at a single locus. Here we use ligation-mediated PCR to analyze DNA cleavage intermediates in thymocytes from mice with targeted RSS mutations at the endogenous TCRβ locus. We show that DNA cleavage does not occur at individual RSSs but rather must be coordinated between RSS pairs flanking gene segments that ultimately form coding joins. Coordination of the DNA cleavage step occurs over great distances in the chromosome and favors intra- over interchromosomal recombination. Furthermore, through several restrictions imposed on the generation of both nonpaired and paired DNA DSBs, this requirement promotes antigen receptor gene integrity and genomic stability in developing lymphocytes undergoing V(D)J recombination.
Slowed Relaxation in Fatigued Skeletal Muscle Fibers ofXenopusand Mouse
Authors: Westerblad Håkan , Lännergren Jan , Allen David G.
Published: July 2002

Slowing of relaxation is an important characteristic of skeletal muscle fatigue. The aim of the present study was to quantify the relative contribution of altered Ca2+ handling (calcium component) and factors down-stream to Ca2+ (cross-bridge component) to the slowing of relaxation in fatigued fibers of Xenopus and mouse. Two types of Xenopus fibers were used: easily fatigued, type 1 fibers and fatigue resistant, type 2 fibers. In these Xenopus fibers the free myoplasmic [Ca2+] ([Ca2+]i) was measured with indo-1, and the relaxation of Ca2+-derived force, constructed from tetanic [Ca2+]i records and in vivo [Ca2+]i-force curves, was analyzed. An alternative method was used in both Xenopus and mouse fibers: fibers were rapidly shortened during the initial phase of relaxation, and the time to the peak of force redevelopment was measured. These two methods gave similar results and showed proportional slowing of the calcium and cross-bridge components of relaxation in both fatigued type 1 and type 2 Xenopus fibers, whereas only the cross-bridge component was slowed in fatigued mouse fibers. Ca2+ removal from the myoplasm during relaxation was markedly less effective in Xenopus fibers as compared to mouse fibers. Fatigued Xenopus fibers displayed a reduced rate of sarcoplasmic reticulum Ca2+ uptake and increased sarcoplasmic reticulum Ca2+ leak. Some fibers were stretched at various times during relaxation. The resistance to these stretches was increased during fatigue, especially in Xenopus fibers, which indicates that longitudinal movements during relaxation had become less pronounced and this might contribute to the increased cross-bridge component of relaxation in fatigue. In conclusion, slowing of relaxation in fatigued Xenopus fibers is caused by impaired Ca2+ handling and altered cross-bridge kinetics, whereas the slowing in mouse fibers is only due to altered cross-bridge kinetics.
Inositol 1,4,5-Trisphosphate (InsP3) and Calcium Interact to Increase the Dynamic Range of InsP3 Receptor-dependent Calcium Signaling
Authors: Kaftan Edward J. , Ehrlich Barbara E. , Watras James
Published: July 2002

The inositol 1,4,5-trisphosphate (InsP3)-gated Ca channel in cerebellum is tightly regulated by Ca (Bezprozvanny, I., J. Watras, and B.E. Ehrlich. 1991. Nature (Lond.). 351:751–754; Finch, E.A., T.J. Turner, and S.M. Goldin. 1991. Science (Wash. DC). 252:443–446; Hannaert-Merah, Z., J.F. Coquil, L. Combettes, M. Claret, J.P. Mauger, and P. Champeil. 1994. J. Biol. Chem. 269:29642–29649; Iino, M. 1990. J. Gen. Physiol. 95:1103–1122; Marshall, I., and C. Taylor. 1994. Biochem. J. 301:591–598). In previous single channel studies, the Ca dependence of channel activity, monitored at 2 μM InsP3, was described by a bell-shaped curve (Bezprozvanny, I., J. Watras, and B.E. Ehrlich. 1991. Nature (Lond.). 351:751–754). We report here that, when we used lower InsP3 concentrations, the peak of the Ca-dependence curve shifted to lower Ca concentrations. Unexpectedly, when we used high InsP3 concentrations, channel activity persisted at Ca concentrations as high as 30 μM. To explore this unexpected response of the channel, we measured InsP3 binding over a broad range of InsP3 concentrations. We found the well-characterized high affinity InsP3 binding sites (with Kd < 1 and 50 nM) (Maeda, N., M. Niinobe, and K. Mikoshiba. 1990. EMBO (Eur. Mol. Biol. Organ.) J. 9:61–67; Mignery, G., T.C. Sudhof, K. Takei, and P. De Camilli. 1989. Nature (Lond.). 342:192–195; Ross, C.A., J. Meldolesi, T.A. Milner, T. Satoh, S. Supattapone, and S.H. Snyder. 1989. Nature (Lond.). 339:468–470) and a low affinity InsP3 binding site (Kd = 10 μM). Using these InsP3 binding sites, we developed a new model that accounts for the shift in the Ca-dependence curve at low InsP3 levels and the maintained channel activity at high Ca and InsP3 levels. The observed Ca dependence of the InsP3-gated Ca channel allows the cell to abbreviate the rise of intracellular Ca in the presence of low levels of InsP3, but also provides a means of maintaining high intracellular Ca during periods of prolonged stimulation.
Inactivation Gating of Kv4 Potassium Channels
Authors: Jerng Henry H. , Shahidullah Mohammad , Covarrubias Manuel
Published: July 2002

Kv4 channels represent the main class of brain A-type K+ channels that operate in the subthreshold range of membrane potentials (Serodio, P., E. Vega-Saenz de Miera, and B. Rudy. 1996. J. Neurophysiol. 75:2174– 2179), and their function depends critically on inactivation gating. A previous study suggested that the cytoplasmic NH2- and COOH-terminal domains of Kv4.1 channels act in concert to determine the fast phase of the complex time course of macroscopic inactivation (Jerng, H.H., and M. Covarrubias. 1997. Biophys. J. 72:163–174). To investigate the structural basis of slow inactivation gating of these channels, we examined internal residues that may affect the mutually exclusive relationship between inactivation and closed-state blockade by 4-aminopyridine (4-AP) (Campbell, D.L., Y. Qu, R.L. Rasmussen, and H.C. Strauss. 1993. J. Gen. Physiol. 101:603–626; Shieh, C.-C., and G.E. Kirsch. 1994. Biophys. J. 67:2316–2325). A double mutation V[404,406]I in the distal section of the S6 region of the protein drastically slowed channel inactivation and deactivation, and significantly reduced the blockade by 4-AP. In addition, recovery from inactivation was slightly faster, but the pore properties were not significantly affected. Consistent with a more stable open state and disrupted closed state inactivation, V[404,406]I also caused hyperpolarizing and depolarizing shifts of the peak conductance–voltage curve (∼5 mV) and the prepulse inactivation curve (>10 mV), respectively. By contrast, the analogous mutations (V[556,558]I) in a K+ channel that undergoes N- and C-type inactivation (Kv1.4) did not affect macroscopic inactivation but dramatically slowed deactivation and recovery from inactivation, and eliminated open-channel blockade by 4-AP. Mutation of a Kv4-specifc residue in the S4–S5 loop (C322S) of Kv4.1 also altered gating and 4-AP sensitivity in a manner that closely resembles the effects of V[404,406]I. However, this mutant did not exhibit disrupted closed state inactivation. A kinetic model that assumes coupling between channel closing and inactivation at depolarized membrane potentials accounts for the results. We propose that components of the pore's internal vestibule control both closing and inactivation in Kv4 K+ channels.
Reactive Oxygen Species Regulate Oxygen-Sensitive Potassium Flux in Rainbow Trout Erythrocytes
Authors: Bogdanova Anna Yu , Nikinmaa Mikko
Published: July 2002

In the present study, we have investigated if reactive oxygen species are involved in the oxygen-dependent regulation of potassium-chloride cotransport activity in trout erythrocyte membrane. An increase in the oxygen level caused an increase in chloride-sensitive potassium transport (K+-Cl− cotransport). 5 mM hydrogen peroxide caused an increase in K+-Cl− cotransport at 5% oxygen. The increase in flux could be inhibited by adding extracellular catalase in the incubation. Pretreatment of the cells with mercaptopropionyl glycine (MPG), a scavenger of reactive oxygen species showing preference for hydroxyl radicals, abolished the activation of the K+-Cl− cotransporter by increased oxygen levels. The inhibition by MPG was reversible, and MPG could not inhibit the activation of transporter by the sulfhydryl reagent, N-ethylmaleimide, indicating that the effect of MPG was due to the scavenging of reactive oxygen species and not to the reaction of MPG with the cotransporter. Copper ions, which catalyze the production of hydroxyl radicals in the Fenton reaction, activated K+-Cl− cotransport significantly at hypoxic conditions (1% O2). These data suggest that hydroxyl radicals, formed from O2 in close vicinity to the cell membrane, play an important role in the oxygen-dependent activation of the K+-Cl− cotransporter.
Development and validation of a monoclonal based immunoassay for the measurement of fungal alpha-amylase: focus on peak exposures
Authors: Elms J. , Denniss S. , Smith M. , Evans P. G. , Wiley K. , Griffin P. , Curran A. D.
Published: July 2002

The inhalation of flour dust has been implicated in the induction of sensitisation and elicitation of respiratory symptoms, such as asthma in bakers. In addition to the cereal allergens present in wheat flour, enzymes in flour improvers, in particular fungal alpha-amylase, are now known to be a significant cause of respiratory allergy in the baking industry.

A monoclonal antibody based enzyme-linked immunoassay (ELISA) was developed using two monoclonal antibodies that recognised two distinct epitopes of the fungal alpha-amylase enzyme. The ELISA had an inter-assay variation of 12.0% at 1360 pg/ml and 12.8% at 564 pg/ml and intra-assay variation of 4.9% at 1340 pg/ml and 6.1% at 504 pg/ml. The assay had a sensitivity of 200 pg/ml. Competitive inhibition assays confirmed that the monoclonal antibodies had no cross reactivity with other enzymes used in the baking industry and could distinguish added fungal alpha-amylase from cereal amylase.

We assessed the levels of exposure to dust, total protein and fungal alpha-amylase in four UK bakeries ranging in size and technical capabilities. Within the bakeries we surveyed, workers were exposed to variable levels of inhalable dust (0.8–39.8 mg/m3), total protein (0–5.7 mg/m3) and fungal alpha-amylase (0–29.8 ng/m3). Consecutive 15 min personal samples taken over a 1 h period demonstrated that the ELISA could measure fungal alpha-amylase exposure in such a 15 min period. Short term peak exposures to fungal alpha-amylase could be identified which may contribute to the sensitisation in individuals who appear to have low exposure levels if measured over a full shift period.
Neural network schemes for detecting rare events in human

genomic DNA
Authors: Choe Wooyoung , Ersoy Okan K. , Bina Minou
Published: July 2002

Motivation: Many problems in molecular biology as well as other areas involve detection of rare events in unbalanced data. We develop two sample stratification schemes in conjunction with neural networks for rare event detection in such databases. Sample stratification is a technique for making each class in a sample have equal influence on decision making. The first scheme proposed stratifies a sample by adding up the weighted sum of the derivatives during the backward pass of training. The second scheme proposed uses a technique of modified bootstrap aggregating. After training neural networks with multiple sets of bootstrapped examples of the rare event classes and subsampled examples of common event classes, multiple voting for classification is performed.

Results: These two schemes make rare event classes have a better chance of being included in the sample used for training neural networks and thus improve the classification accuracy for rare event detection. The experimental performance of the two schemes using two sets of human DNA sequences as well as another set of Gaussian data indicates that proposed schemes have the potential of significantly improving accuracy of neural networks to recognize rare events.

Availability: Web service is at http://128.46.200.193/sharing/bioinfo.

Contact: wooyoung@ecn.purdue.edu; ersoy@ecn.purdue.edu
Cognitive therapy for depression
Authors: Scott Jan
Published: July 2002

There is considerable empirical support for the use of cognitive therapy in the treatment of mild to moderately severe acute major depression. More recent research has focused on the utility of this approach in severe or chronic depressive disorders, in relapse prevention and also on the potential benefits of combining cognitive therapy with medication. This paper attempts to clarify the empirical data on these important issues in order to identify further the role of cognitive therapy in day-to-day clinical practice. It also provides an overview of findings regarding predictors of response to cognitive therapy and the possible mediators of its effects.
FEDERAL LAND EXCHANGES: 1960–1999
Authors: Espey Molly
Published: July 2002

This research estimates the influence of various economic, legal, and political factors on the occurrence of federal land exchanges over time. Economic growth is found to significantly increase the number of land exchanges. The passage of laws related to exchanges and the political affiliation of the president also influence the number of exchanges. Finally, appropriations for land acquisition are inversely related to the number of federal land exchanges.
Reconciling Individual and Aggregate Evidence Concerning Partisan Stability: Applying Time-Series Models to Panel Survey Data
Authors: Green Donald P. , Yoon David H.
Published: July 2002

Party identification has been studied extensively using both individual- and aggregate-level data. This paper attempts to formulate a statistical model that can account for the range of empirical generalizations that have emerged from aggregate time series and panel surveys. Using Monte Carlo simulation, we show that only certain types of data generation processes can account for these empirical regularities. Deciding which of the remaining types best explains the data means investigating the ways in which individual-level partisanship behaves over time. Partisanship at the aggregate-level tends to be highly autocorrelated, reequilibrating slowly in the wake of each perturbation. Working downward from the analysis of aggregate data, previous researchers argued that aggregate partisanship is fractionally integrated and contended that dynamics at the individual level are therefore heterogeneous. Using data from three panel surveys, we present the first direct assessment of individual-level dynamics. We also investigate the hypothesis that these dynamics vary among individuals, a claim that motivates much recent work on fractionally integrated time series. The model that best explains the observed characteristics of party identification is one in which individuals respond in similar ways to external shocks, reequilibrate rapidly thereafter, and seldom change their equilibrium level of partisan attachment.
CAG--polyglutamine repeat mutations: independence from gene context
Authors: Ordway Jared M. , Cearley Jamie A. , Detloff Peter J.
Published: July 2002

Several neurological disorders have been attributed to the inheritance of long CAG–polyglutamine repeats. Unlike classical mutations, whose deleterious effects are totally dependent on the context of the gene in which they reside, these translated CAG repeat mutations have been shown to cause neurotoxicity and neuronal intranuclear inclusions when expressed outside their natural gene context. We provide a description of mice with different lengths of repeat in the foreign context of the murine
Hprt
locus, focusing on aspects of the phenotype that provide an insight into the mechanism by which this unusual mutation might cause toxicity.
The omnivorous Tyrolean Iceman: colon contents (meat, cereals, pollen, moss and whipworm) and stable isotope analyses
Authors: Dickson James H. , Oeggl Klaus , Holden Timothy G. , Handley Linda L. , O'Connell Tamsin C. , Preston Thomas
Published: July 2002

The contents of the colon of the Tyrolean Iceman who livedca.5300 years ago include muscle fibres, cereal remains, a diversity of pollen, and most notably that of the hop hornbeam (Ostrya carpinifolia) retaining cellular contents, as well as a moss leaf (Neckera complanata) and eggs of the parasitic whipworm (Trichuris trichiura). Based almost solely on stable isotope analyses and ignoring the work on the colon contents, two recently published papers on the Iceman's diet draw ill-founded conclusions about vegetarianism and even veganism. Neither the pollen nor the moss is likely to have been deliberately consumed as food by the Iceman. All the available evidence concerning the Iceman's broad-based diet is reviewed and the significance of the colon contents for matters other than assessment of food intake is outlined.
PIASy, a nuclear matrix–associated SUMO E3 ligase, represses LEF1 activity by sequestration into nuclear bodies
Authors: Sachdev Shrikesh , Bruhn Laurakay , Sieber Heidemarie , Pichler Andrea , Melchior Frauke , Grosschedl Rudolf
Published: July 2002

The Wnt-responsive transcription factor LEF1 can activate transcription in association with β-catenin and repress transcription in association with Groucho. In search of additional regulatory mechanisms of LEF1 function, we identified the protein inhibitor of activated STAT, PIASy, as a novel interaction partner of LEF1. Coexpression of PIASy with LEF1 results in potent repression of LEF1 activity and in covalent modification of LEF1 with SUMO. PIASy markedly stimulates the sumoylation of LEF1 and multiple other proteins in vivo and functions as a SUMO E3 ligase for LEF1 in a reconstituted system in vitro. Moreover, PIASy binds to nuclear matrix–associated DNA sequences and targets LEF1 to nuclear bodies, suggesting that PIASy-mediated subnuclear sequestration accounts for the repression of LEF1 activity.
Specific protein dynamics near the solvent glass transition assayed by radiation‐induced structural changes
Authors: Weik M. , Ravelli R.B.G. , Silman I. , Sussman J.L. , Gros P. , Kroon J.
Published: July 2002

The nature of the dynamical coupling between a protein and its surrounding solvent is an important, yet open issue. Here we used temperature‐dependent protein crystallography to study structural alterations that arise in the enzyme acetylcholinesterase upon X‐ray irradiation at two temperatures: below and above the glass transition of the crystal solvent. A buried disulfide bond, a buried cysteine, and solvent exposed methionine residues show drastically increased radiation damage at 155 K, in comparison to 100 K. Additionally, the irradiation‐induced unit cell volume increase is linear at 100 K, but not at 155 K, which is attributed to the increased solvent mobility at 155 K. Most importantly, we observed conformational changes in the catalytic triad at the active site at 155 K but not at 100 K. These changes lead to an inactive catalytic triad conformation and represent, therefore, the observation of radiation‐inactivation of an enzyme at the atomic level. Our results show that at 155 K, the protein has acquired—at least locally—sufficient conformational flexibility to adapt to irradiation‐induced alterations in the conformational energy landscape. The increased protein flexibility may be a direct consequence of the solvent glass transition, which expresses as dynamical changes in the enzyme's environment. Our results reveal the importance of protein and solvent dynamics in specific radiation damage to biological macromolecules, which in turn can serve as a tool to study protein flexibility and its relation to changes in a protein's environment.
When do bubbles cause a floating body to sink?
Authors: Denardo Bruce , Pringle Leonard , DeGrace Carl , McGuire Michael
Published: July 2002

The introduction of gas bubbles into a liquid might be expected to cause a relatively large floating body to sink when the average density of the fluid is less than the average density of the body. However, the rising bubbles could entrain the liquid, which would produce an upward drag on the body and thus lower the fluid density required to sink the body. Due to this drag, it is in fact not clear whether any amount of bubbles can cause a floating body to sink. In addition, there are many other effects that could alter the requisite fluid density. We describe qualitative lecture demonstrations which show that bubbles can indeed sink a body, including the case of ice in water. We also describe a quantitative experiment to determine the density of bubbly water required to sink a spherical body. The specific gravity of this body is varied from 0.99 to 0.75, and the results are compared to a simple theory.
Fluctuations in the number of particles of the ideal gas: A simple example of explicit finite-size effects
Authors: Román F. L. , González A. , White J. A. , Velasco S.
Published: July 2002

The fluctuations in the number of particles of the ideal gas are calculated using the canonical and the grand canonical ensembles. The two results differ by a factor which accounts for the relative size of the total volume and the subvolume where the canonical ensemble fluctuations are calculated. This factor gives a simple example of explicit finite-size effects, because it arises from considering a fixed number of particles in the canonical ensemble. Our simulation results for the isothermal compressibility of the hard disk fluid with a finite number of particles are improved by applying this correction.
Illustration of the Fermi–Dirac statistics
Authors: Arnaud J. , Boé J. M. , Chusseau L. , Philippe F.
Published: July 2002

The distribution of electrons in small one-dimensional systems is obtained under the assumption of evenly spaced energy levels. The method consists of considering isolated systems and shifting electrons from their zero-temperature location. The distribution is then expressed in terms of the number of partitions of integers. When the system is in thermal contact with an electrical insulator, the electron distribution is obtained by averaging the previous result with the Boltzmann factor as a weight. Finally, when the system is in thermal and electrical contact with a large medium, the Fermi–Dirac distribution emerges through averaging over the number N of electrons. The statistics of light emitted or absorbed by the electron gas is obtained without quantization of the optical field. Our rigorous though elementary treatment helps clarify concepts employed in statistical mechanics.
Temperature dependence of ultrasonic enhancement with a site-targeted contrast agent
Authors: Hall Christopher S. , Marsh Jon N. , Scott Michael J. , Gaffney Patrick J. , Wickline Samuel A. , Lanza Gregory M.
Published: July 2002

Molecular imaging contrast agents specifically detect the biochemical “signatures” of disease before anatomical manifestations are apparent. Sensitive and specific localization of fibrin both in vivo and in vitro has been demonstrated with the use of a ligand-directed liquid perfluorocarbon nanoparticle. Since the acoustic properties of perfluorocarbons are known to vary with temperature, it was hypothesized that temperature could be used to augment the magnitude of enhancement imparted by targeted nanoparticles. Accordingly, the acoustic backscatter of two different substrates, nitrocellulose membrane and human plasma clot, targeted by the nanoparticles was measured at temperatures ranging from 27° to 47 °C in 5 °C increments. Classic avidin–biotin interactions were utilized to couple biotinylated nanoparticles to avidin-conjugated nitrocellulose membranes. Ultrasonic contrast enhancement of the nitrocellulose membrane at 25 MHz, measured by acoustic microscopy, increased from 2.0±0.3 dB at 27 °C to 3.7±0.4 at 47 °C. In a similar experiment, antifibrin nanoparticles bound to human plasma clots also exhibited temperature-dependent ultrasonic signal enhancement ranging from 13.9±1.5 dB at 27 °C to 18.1±1.5 dB at 47 °C. The increase in ultrasonic contrast enhancement measured was well described by a simple, acoustic transmission line model with temperature-dependent impedance. These results suggest that temperature-dependent changes in acoustic backscatter may be used to further differentiate tissues targeted with site-specific nanoparticles from surrounding normal soft tissues.
Passive ranging errors due to multipath distortion of deterministic transient signals with application to the localization of small arms fire
Authors: Ferguson Brian G. , Lo Kam W.
Published: July 2002

A passive ranging technique based on wavefront curvature is used to estimate the ranges and bearings of impulsive sound sources represented by small arms fire. The discharge of a firearm results in the generation of a transient acoustic signal whose energy propagates radially outwards from the omnidirectional source. The radius of curvature of the spherical wavefront at any instant is equal to the instantaneous range from the source. The curvature of the acoustic wavefront is sensed with a three-microphone linear array by first estimating the differential time of arrival (or time delay) of the acoustic wavefront at each of the two adjacent sensor pairs and then processing the time-delay information to extract the range and bearing of the source. However, modeling the passive ranging performance of the wavefront curvature method for a deterministic transient signal source in a multipath environment shows that when the multipath and direct path arrivals are unresolvable, the time-delay estimates are biased which, in turn, biases the range estimates. The model explains the observed under-ranging of small arms firing positions during a field experiment.
Experimental identification of finite cylindrical shell vibration modes
Authors: Haumesser Lionel , Décultot Dominique , Léon Fernand , Maze Gérard
Published: July 2002

Acoustic scattering from a finite air-filled elastic cylindrical shell, immersed in water, is investigated. The shell is made of stainless steel and has a thickness to outer radius ratio of 17%. The considered dimensionless frequency range extends over 7⩽k1a⩽22 (k1: wave number in water, a: outer radius). Bistatic measurements are carried out to identify vibration modes related to the phase matching of the first guided wave, T0, propagating on the shell. Both transducers, the emitter and the receiver, are positioned at the same angular distance with regard to the normal axis of the shell. The emitter transducer is fixed at a given position. In order to identify circumferential modes of vibration, the receiver transducer is made to rotate in the azimuthal plane, normal to the shell axis. Results obtained are plotted in functions of dimensionless frequency and azimuthal angle. Vibration modes along the shell’s length are identified by moving the receiver transducer parallel to the shell axis. In this case, results are plotted in functions of dimensionless frequency and axial wave number. The experimental investigation is corroborated by theoretical results obtained from approximate calculations for thick finite cylindrical shells [Scot F. Morse et al., J. Acoust. Soc. Am. 103, 785–794 (1998)]. The evolution of the mode position with respect to the incidence angle is discussed so as to clarify peak patterns in backscattered resonance spectra.
Status of road traffic noise in Calcutta metropolis, India
Authors: Chakrabarty Debashis , Chandra Santra Subhas , Mukherjee Abirlal , Roy Biva , Das Prasun
Published: July 2002

Road traffic noise was measured at 24 traffic junctions of Calcutta, India during the summer of 1993, with the simultaneous recording of the number of vehicles passing. The A-weighted values of Leq(24), Ldn, L10, L90, L99, Lnp and TNI were determined. Statistical regression equations of Leq(h) were developed as a function of logarithm of the equivalent number of light/heavy vehicles per hour for the entire city and for zones within the city.