bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of cyclosporin A on inflammatory cytokine production by U937 monocyte‐like cells
Authors: Garcia Juan E. Losa , de Cabo María Rosa Martín , Rodríguez Fernando Mateos , Losada Jesús Pérez , López Antonio Jiménez , Arellano José Luis Pérez
Published: July 2002

Cyclosporin A (CsA) is an immunosuppresor drug that has been used in the treatment of several types of inflammatory diseases. In some of them the inhibition of T‐lymphocyte activation does not suitably account for the observed beneficial effect, suggesting that CsA could act on other types of cells. The present study was undertaken to determine the effect of CsA on inflammatory cytokine secretion by U937 monocyte cells. Undifferentiated and dimethylsulfoxide (DMSO) differentiated U937 cells were incubated with different concentrations of CsA (200, 20 and 2 ng/mL) in the presence or absence of phorbol‐myristateacetate (PMA). Interleukin‐1g (IL‐1β), tumor necrosis factor‐α (TNF‐α), IL‐6 and IL‐8 levels were measured in supernatants using specific enzyme‐linked immunosorbent assays. At the highest concentration used (200 ng/mL) CsA decreased the basal and stimulated secretion of all the inflammatory cytokines studied in both undifferentiated and differentiated cells, with the only exception of PMA‐stim ulated IL‐1 secretion by undifferentiated cells. However, only basal secretion of interleukin‐8 in both undifferentiated and DMSO‐differentiated U937 cells was significantly reduced by CsA at the highest concentration (200 ng/ mL). At therapeutic concentrations in vivo, CsA exerts a predominant effect on IL‐8 secretion by human mononuclear phagocytes.
Molecular Neurobiology of Addiction
Authors: Nestler Eric J.
Published: July 2002

Addiction can be viewed as a form of drug‐induced neural plasticity. One of the best established molecular mechanisms of addiction is the upregulation of the cAMP second messenger pathway, which occurs in many neuronal cell types in response to chronic administration of opiates or other drugs of abuse. This upregulation and the resulting activation of the transcription factor CREB appear to mediate aspects of tolerance and dependence. In contrast, induction of another transcription factor, termed ΔFosB, exerts the opposite effect and may contribute to sensitized responses to drug exposure. Knowledge of these mechanisms could lead to more effective treatments for addictive disorders.
Toxicity of 2,4,6-TrinitrotoIuene (TNT) in Hispid Cotton Rats (Sigmodon hispidus): Hematological, Biochemical, and Pathological Effects
Authors: Reddy Gunda , Chandra Sundeep A. M. , Lish James W. , Quails Charles W.
Published: July 2002

The contamination of soil and water with munitions chemicals and their degradation products has been reported at certain munitions production waste disposal sites and at certain Army installations. The effects of 2,4,6-trinitrotoluene (TNT) on wild cotton rats ( Sigmodon hispidus) were evaluated to identify target organ toxicity that could be used to develop biomarkers for exposure assessment for ecological and health risks. The oral LD50values for TNT in corn oil were 607 and 767 mg/kg body weights for male and female cotton rats respectively. Hematological, pathological, and biochemical effects of TNT were determined after daily oral gavage of TNT in corn oil at doses of 0, 75.9, 151.8, and 303.5 (males) or 0, 96, 192, and 384 mg/kg (females) for 7 days. Cotton rats treated with TNT showed an increase in spleen weights in males (303.5 mg/kg) and in females (192 and 384 mg/kg). Cotton rats of both sexes treated for 7 days with TNT had marked hemolytic anemia with reduced erythrocytes, hemoglobin, and hematocrit in high-dose groups; methemoglobin levels were elevated significantly in males at mid and high dose. Hepatic drug metabolizing enzyme analysis revealed that microsomal O-dealkylation of methoxy, ethoxy, and pentoxy resorufin were elevated in male (high dose) cotton rats. The activity of hepatic glutathione S-transferases (GST) was significantly elevated in male (mid and high dose) and female (all doses) cotton rats exposed to TNT. Histopathological analysis of spleen revealed mild to marked splenic congestion with mild extramedullary hematopoiesis, hemosiderosis, and lymphoid hyperplasia in male and female cotton rats treated with TNT (all doses). Liver weights were increased in males (mid and high dose) and in females (high-dose group). In the high-dose groups, histological changes in liver (mild to moderate hepatocellular hypertrophy, increased hemosiderin pigment in Kupffer cells) in both sexes, and in testis (premature exfoliation of spermatozoa from dilated seminiferous tubules) were observed (mid and high dose). These results suggest that hepatic GST and hemolytic anemia may be biomarkers in cotton rats of terrestrial contamination with TNT or other nitroaromatic explosive compounds.
Consensus and variation between speech and language therapists in the assessment and selection of preschool children for intervention: a body of knowledge or idiosyncratic decisions?
Authors: Roulstone Sue
Published: July 2002

The aim was to investigate consensus between speech and language therapists (SLT) during the initial assessment of preschool children. A multimethod design was used. The qualitative component provided SLT's own views of the process as the starting point for the study. Data were generated from semistructured interviews and focus group discussions. The interviews investigated factors considered during an initial assessment of a preschool child. The focus groups discussed the process and content of a videotaped initial assessment carried out by the author. Quantitative data was generated from six case analyses and from a card sorting exercise. In the former, SLT were asked to record important features of newly referred cases. In the latter, SLT sorted statements about preschool children according to a simple concern/not concerned decision. Data collection tasks and analysis took place successively to build a composite and triangulated picture. The main subjects were 11 SLT, experienced in childrens' work with a further six acting as a broader reference group. The results show an interacting pattern of consensus and variation. SLT agreed about the relative priority of children, categories that informed the assessment; they made similar interpretations of incoming information. There was variation in their preferred assessment methods, in the thresholds of concern and in the interventions offered. Sources of variation that were identified included individual confidence in and access to assessments, personal levels of concern and in the approach to the SLT's role, as well as the influence of workplace policies. The study concluded that, consensus and variation both exist within the context of SLT's initial assessment of preschool children, but that there are discernible patterns to the variation. While such variation may be inevitable, it is suggested that any variation should be made explicit and used to challenge and develop the shared knowledge base of the speech and language therapy profession.
PDGF, TGF-β, and Heterotypic Cell–Cell Interactions Mediate Endothelial Cell–induced Recruitment of 10T1/2 Cells and Their Differentiation to a Smooth Muscle Fate
Authors: Hirschi Karen K. , Rohovsky Stephanie A. , D'Amore Patricia A.
Published: July 2002

We aimed to determine if and how endothelial cells (EC) recruit precursors of smooth muscle cells and pericytes and induce their differentiation during vessel formation. Multipotent embryonic 10T1/2 cells were used as presumptive mural cell precursors. In an under-agarose coculture, EC induced migration of 10T1/2 cells via platelet-derived growth factor BB. 10T1/2 cells in coculture with EC changed from polygonal to spindle-shaped, reminiscent of smooth muscle cells in culture. Immunohistochemical and Western blot analyses were used to examine the expression of smooth muscle (SM)-specific markers in 10T1/2 cells cultured in the absence and presence of EC. SM-myosin, SM22α, and calponin proteins were undetectable in 10T1/2 cells cultured alone; however, expression of all three SM-specific proteins was significantly induced in 10T1/2 cells cocultured with EC. Treatment of 10T1/2 cells with TGF-β induced phenotypic changes and changes in SM markers similar to those seen in the cocultures. Neutralization of TGF-β in the cocultures blocked expression of the SM markers and the shape change. To assess the ability of 10T1/2 cells to contribute to the developing vessel wall in vivo, prelabeled 10T1/2 cells were grown in a collagen matrix and implanted subcutaneously into mice. The fluorescently marked cells became incorporated into the medial layer of developing vessels where they expressed SM markers. These in vitro and in vivo observations shed light on the cell–cell interactions that occur during vessel development, as well as in pathologies in which developmental processes are recapitulated.
Rho GTPases Control Polarity, Protrusion, and Adhesion during Cell Movement
Authors: Nobes Catherine D. , Hall Alan
Published: July 2002

Cell movement is essential during embryogenesis to establish tissue patterns and to drive morphogenetic pathways and in the adult for tissue repair and to direct cells to sites of infection. Animal cells move by crawling and the driving force is derived primarily from the coordinated assembly and disassembly of actin filaments. The small GTPases, Rho, Rac, and Cdc42, regulate the organization of actin filaments and we have analyzed their contributions to the movement of primary embryo fibroblasts in an in vitro wound healing assay. Rac is essential for the protrusion of lamellipodia and for forward movement. Cdc42 is required to maintain cell polarity, which includes the localization of lamellipodial activity to the leading edge and the reorientation of the Golgi apparatus in the direction of movement. Rho is required to maintain cell adhesion during movement, but stress fibers and focal adhesions are not required. Finally, Ras regulates focal adhesion and stress fiber turnover and this is essential for cell movement. We conclude that the signal transduction pathways controlled by the four small GTPases, Rho, Rac, Cdc42, and Ras, cooperate to promote cell movement.
Cd38/Adp-Ribosyl Cyclase
Authors: Sun Li , Adebanjo Olugbenga A. , Moonga Baljit S. , Corisdeo Susanne , Anandatheerthavarada Hindupur K. , Biswas Gopa , Arakawa Toshiya , Hakeda Yoshiyuki , Koval Antoliy , Sodam Bali , Bevis Peter J.R. , Moser A. James , Lai F. Anthony , Epstein Solomon , Troen Bruce R. , Kumegawa Masayoshi , Zaidi Mone
Published: July 2002

The multifunctional ADP-ribosyl cyclase, CD38, catalyzes the cyclization of NAD+ to cyclic ADP-ribose (cADPr). The latter gates Ca2+ release through microsomal membrane-resident ryanodine receptors (RyRs). We first cloned and sequenced full-length CD38 cDNA from a rabbit osteoclast cDNA library. The predicted amino acid sequence displayed 59, 59, and 50% similarity, respectively, to the mouse, rat, and human CD38. In situ RT-PCR revealed intense cytoplasmic staining of osteoclasts, confirming CD38 mRNA expression. Both confocal microscopy and Western blotting confirmed the plasma membrane localization of the CD38 protein. The ADP-ribosyl cyclase activity of osteoclastic CD38 was next demonstrated by its ability to cyclize the NAD+ surrogate, NGD+, to its fluorescent derivative cGDP-ribose. We then examined the effects of CD38 on osteoclast function. CD38 activation by an agonist antibody (A10) in the presence of substrate (NAD+) triggered a cytosolic Ca2+ signal. Both ryanodine receptor modulators, ryanodine, and caffeine, markedly attenuated this cytosolic Ca2+ change. Furthermore, the anti-CD38 agonist antibody expectedly inhibited bone resorption in the pit assay and elevated interleukin-6 (IL-6) secretion. IL-6, in turn, enhanced CD38 mRNA expression. Taken together, the results provide compelling evidence for a new role for CD38/ADP-ribosyl cyclase in the control of bone resorption, most likely exerted via cADPr.
Tumor Suppressor P53 Binding Protein 1 (53bp1) Is Involved in DNA Damage–Signaling Pathways
Authors: Rappold Irene , Iwabuchi Kuniyoshi , Date Takayasu , Chen Junjie
Published: July 2002

The tumor suppressor p53 binding protein 1 (53BP1) binds to the DNA-binding domain of p53 and enhances p53-mediated transcriptional activation. 53BP1 contains two breast cancer susceptibility gene 1 COOH terminus (BRCT) motifs, which are present in several proteins involved in DNA repair and/or DNA damage–signaling pathways. Thus, we investigated the potential role of 53BP1 in DNA damage–signaling pathways. Here, we report that 53BP1 becomes hyperphosphorylated and forms discrete nuclear foci in response to DNA damage. These foci colocalize at all time points with phosphorylated H2AX (γ-H2AX), which has been previously demonstrated to localize at sites of DNA strand breaks. 53BP1 foci formation is not restricted to γ-radiation but is also detected in response to UV radiation as well as hydroxyurea, camptothecin, etoposide, and methylmethanesulfonate treatment. Several observations suggest that 53BP1 is regulated by ataxia telangiectasia mutated (ATM) after DNA damage. First, ATM-deficient cells show no 53BP1 hyperphosphorylation and reduced 53BP1 foci formation in response to γ-radiation compared with cells expressing wild-type ATM. Second, wortmannin treatment strongly inhibits γ-radiation–induced hyperphosphorylation and foci formation of 53BP1. Third, 53BP1 is readily phosphorylated by ATM in vitro. Taken together, these results suggest that 53BP1 is an ATM substrate that is involved early in the DNA damage–signaling pathways in mammalian cells.
Wnt signaling promotes oncogenic transformation by inhibiting c-Myc–induced apoptosis
Authors: You Zongbing , Saims Daniel , Chen Shaoqiong , Zhang Zhaocheng , Guttridge Denis C. , Guan Kun-liang , MacDougald Ormond A. , Brown Anthony M.C. , Evan Gerard , Kitajewski Jan , Wang Cun-Yu
Published: July 2002

Aberrant activation of the Wnt/β-catenin signaling pathway is associated with numerous human cancers and often correlates with the overexpression or amplification of the c-myc oncogene. Paradoxical to the cellular transformation potential of c-Myc is its ability to also induce apoptosis. Using an inducible c-MycER expression system, we found that Wnt/β-catenin signaling suppressed apoptosis by inhibiting c-Myc–induced release of cytochrome c and caspase activation. Both cyclooxygenase 2 and WISP-1 were identified as effectors of the Wnt-mediated antiapoptotic signal. Soft agar assays showed that neither c-Myc nor Wnt-1 alone was sufficient to induce cellular transformation, but that Wnt and c-Myc coordinated in inducing transformation. Furthermore, coexpression of Wnt-1 and c-Myc induced high-frequency and rapid tumor growth in nude mice. Extensive apoptotic bodies were characteristic of c-Myc–induced tumors, but not tumors induced by coactivation of c-Myc and Wnt-1, indicating that the antiapoptotic function of Wnt-1 plays a critical role in the synergetic action between c-Myc and Wnt-1. These results elucidate the molecular mechanisms by which Wnt/β-catenin inhibits apoptosis and provide new insight into Wnt signaling-mediated oncogenesis.
Targeted Deletion of the Lipopolysaccharide (LPS)-binding Protein Gene Leads to Profound Suppression of LPS Responses Ex Vivo, whereas In Vivo Responses Remain Intact
Authors: Wurfel Mark M. , Monks Brian G. , Ingalls Robin R. , Dedrick Russell L. , Delude Russell , Zhou Dahua , Lamping Norbert , Schumann Ralf R. , Thieringer Rolf , Fenton Matthew J. , Wright Samuel D. , Golenbock Douglas
Published: July 2002

Gram-negative bacterial lipopolysaccharide (LPS) stimulates phagocytic leukocytes by interacting with the cell surface protein CD14. Cellular responses to LPS are markedly potentiated by the LPS-binding protein (LBP), a lipid-transfer protein that binds LPS aggregates and transfers LPS monomers to CD14. LBP also transfers LPS to lipoproteins, thereby promoting the neutralization of LPS. LBP present in normal plasma has been shown to enhance the LPS responsiveness of cells in vitro. The role of LBP in promoting LPS responsiveness in vivo was tested in LBP-deficient mice produced by gene targeting in embryonic stem cells. Whole blood from LBP-deficient animals was 1,000-fold less responsive to LPS as assessed by the release of tumor necrosis factor (TNF)-α. Blood from gene-targeted mice was devoid of immunoreactive LBP, essentially incapable of transferring LPS to CD14 in vitro, and failed to support cellular responses to LPS. These activities were restored by the addition of exogenous recombinant murine LBP to the plasma. Despite these striking in vitro findings, no significant differences in TNF-α levels were observed in plasma from wild-type and LBP-deficient mice injected with LPS. These data suggest the presence of an LBP-independent mechanism for responding to LPS. These LBP knockout mice may provide a tool for discovering the nature of the presumed second mechanism for transferring LPS to responsive cells.
Simultaneous Humoral and Cellular Immune Response against Cancer–Testis Antigen NY-ESO-1: Definition of Human Histocompatibility Leukocyte Antigen (HLA)-A2–binding Peptide Epitopes
Authors: Jäger Elke , Chen Yao-Tseng , Drijfhout Jan W. , Karbach Julia , Ringhoffer Mark , Jäger Dirk , Arand Michael , Wada Hisashi , Noguchi Yuji , Stockert Elisabeth , Old Lloyd J. , Knuth Alexander
Published: July 2002

A growing number of human tumor antigens have been described that can be recognized by cytotoxic T lymphocytes (CTLs) in a major histocompatibility complex (MHC) class I–restricted fashion. Serological screening of cDNA expression libraries, SEREX, has recently been shown to provide another route for defining immunogenic human tumor antigens. The detection of antibody responses against known CTL-defined tumor antigens, e.g., MAGE-1 and tyrosinase, raised the question whether antibody and CTL responses against a defined tumor antigen can occur simultaneously in a single patient. In this paper, we report on a melanoma patient with a high-titer antibody response against the “cancer–testis” antigen NY-ESO-1. Concurrently, a strong MHC class I–restricted CTL reactivity against the autologous NY-ESO-1–positive tumor cell line was found. A stable CTL line (NW38-IVS-1) was established from this patient that reacted with autologous melanoma cells and with allogeneic human histocompatibility leukocyte antigen (HLA)-A2−, NY-ESO-1–positive, but not NY-ESO-1–negative, melanoma cells. Screening of NY-ESO-1 transfectants with NW38-IVS-1 revealed NY-ESO-1 as the relevant CTL target presented by HLA-A2. Computer calculation identified 26 peptides with HLA-A2–binding motifs encoded by NY-ESO-1. Of these, three peptides were efficiently recognized by NW38-IVS-1. Thus, we show that antigen-specific humoral and cellular immune responses against human tumor antigens may occur simultaneously. In addition, our analysis provides a general strategy for identifying the CTL-recognizing peptides of tumor antigens initially defined by autologous antibody.
Hematopoietic Progenitor Cell Rolling in Bone Marrow Microvessels: Parallel Contributions by Endothelial Selectins and Vascular Cell Adhesion Molecule 1
Authors: Mazo Irina B. , Gutierrez-Ramos Jose-Carlos , Frenette Paul S. , Hynes Richard O. , Wagner Denisa D. , von Andrian Ulrich H.
Published: July 2002

We have used intravital microscopy to study physiologically perfused microvessels in murine bone marrow (BM). BM sinusoids and venules, but not adjacent bone vessels, supported rolling interactions of hematopoietic progenitor cells. Rolling did not involve L-selectin, but was partially reduced in wild-type mice treated with antibodies to P- or E-selectin and in mice that were deficient in these two selectins. Selectin-independent rolling was mediated by α4 integrins, which interacted with endothelial vascular cell adhesion molecule (VCAM)-1. Parallel contribution of the endothelial selectins and VCAM-1 is not known to direct blood cell trafficking to other noninflamed tissues. This combination of constitutively expressed adhesion molecules may thus constitute a BM-specific recruitment pathway for progenitor cells analogous to the vascular addressins that direct selective lymphocyte homing to lymphoid organs.
Association of the Adaptor Molecule Lat with Cd4 and Cd8 Coreceptors Identifies a New Coreceptor Function in T Cell Receptor Signal Transduction
Authors: Bosselut Rémy , Zhang Weiguo , Ashe Jennifer M. , Kopacz Jeffrey L. , Samelson Lawrence E. , Singer Alfred
Published: July 2002

Linker for activation of T cells (LAT) is an adaptor protein whose tyrosine phosphorylation is critical for transduction of the T cell receptor (TCR) signal. LAT phosphorylation is accomplished by the protein tyrosine kinase ZAP-70, but it is not at all clear how LAT (which is not associated with the TCR) encounters ZAP-70 (which is bound to the TCR). Here we show that LAT associates with surface CD4 and CD8 coreceptors and that its association is promoted by the same coreceptor cysteine motif that mediates Lck binding. In fact, LAT competes with Lck for binding to individual coreceptor molecules but differs from Lck in its preferential association with CD8 rather than CD4 in CD4+CD8+ thymocytes. Importantly, as a consequence of LAT association with surface coreceptors, coengagement of the TCR with surface coreceptors induces LAT phosphorylation and the specific recruitment of downstream signaling mediators to coreceptor-associated LAT molecules. These results point to a new function for CD4 and CD8 coreceptors in TCR signal transduction, namely to promote LAT phosphorylation by ZAP-70 by recruiting LAT to major histocompatibility complex–engaged TCR complexes.
Mutation in the M1 Domain of the Acetylcholine Receptor α Subunit Decreases the Rate of Agonist Dissociation
Authors: Wang Hai-Long , Auerbach Anthony , Bren Nina , Ohno Kinji , Engel Andrew G. , Sine Steven M.
Published: July 2002

We describe the kinetic consequences of the mutation N217K in the M1 domain of the acetylcholine receptor (AChR) α subunit that causes a slow channel congenital myasthenic syndrome (SCCMS). We previously showed that receptors containing αN217K expressed in 293 HEK cells open in prolonged activation episodes strikingly similar to those observed at the SCCMS end plates. Here we use single channel kinetic analysis to show that the prolonged activation episodes result primarily from slowing of the rate of acetylcholine (ACh) dissociation from the binding site. Rate constants for channel opening and closing are also slowed but to much smaller extents. The rate constants derived from kinetic analysis also describe the concentration dependence of receptor activation, revealing a 20-fold shift in the EC50 to lower agonist concentrations for αN217K. The apparent affinity of ACh binding, measured by competition against the rate of 125I-α-bungarotoxin binding, is also enhanced 20-fold by αN217K. Both the slowing of ACh dissociation and enhanced apparent affinity are specific to the lysine substitution, as the glutamine and glutamate substitutions have no effect. Substituting lysine for the equivalent asparagine in the β, ε, or δ subunits does not affect the kinetics of receptor activation or apparent agonist affinity. The results show that a mutation in the amino-terminal portion of the M1 domain produces a localized perturbation that stabilizes agonist bound to the resting state of the AChR.
Gating Kinetics of Single Large-Conductance Ca2+-Activated K+ Channels in High Ca2+ Suggest a Two-Tiered Allosteric Gating Mechanism✪
Authors: Rothberg Brad S. , Magleby Karl L.
Published: July 2002

The Ca2+-dependent gating mechanism of large-conductance calcium-activated K+ (BK) channels from cultured rat skeletal muscle was examined from low (4 μM) to high (1,024 μM) intracellular concentrations of calcium (Ca2+i) using single-channel recording. Open probability (Po) increased with increasing Ca2+i (K0.5 11.2 ± 0.3 μM at +30 mV, Hill coefficient of 3.5 ± 0.3), reaching a maximum of ∼0.97 for Ca2+i ∼ 100 μM. Increasing Ca2+i further to 1,024 μM had little additional effect on either Po or the single-channel kinetics. The channels gated among at least three to four open and four to five closed states at high levels of Ca2+i (>100 μM), compared with three to four open and five to seven closed states at lower Ca2+i. The ability of kinetic schemes to account for the single-channel kinetics was examined with simultaneous maximum likelihood fitting of two-dimensional (2-D) dwell-time distributions obtained from low to high Ca2+i. Kinetic schemes drawn from the 10-state Monod-Wyman-Changeux model could not describe the dwell-time distributions from low to high Ca2+i. Kinetic schemes drawn from Eigen's general model for a ligand-activated tetrameric protein could approximate the dwell-time distributions but not the dependency (correlations) between adjacent intervals at high Ca2+i. However, models drawn from a general 50 state two-tiered scheme, in which there were 25 closed states on the upper tier and 25 open states on the lower tier, could approximate both the dwell-time distributions and the dependency from low to high Ca2+i. In the two-tiered model, the BK channel can open directly from each closed state, and a minimum of five open and five closed states are available for gating at any given Ca2+i. A model that assumed that the apparent Ca2+-binding steps can reach a maximum rate at high Ca2+i could also approximate the gating from low to high Ca2+i. The considered models can serve as working hypotheses for the gating of BK channels.
Ph-Dependent Inhibition of Voltage-Gated H+ Currents in Rat Alveolar Epithelial Cells by Zn2+ and Other Divalent Cations
Authors: Cherny Vladimir V. , DeCoursey Thomas E.
Published: July 2002

Inhibition by polyvalent cations is a defining characteristic of voltage-gated proton channels. The mechanism of this inhibition was studied in rat alveolar epithelial cells using tight-seal voltage clamp techniques. Metal concentrations were corrected for measured binding to buffers. Externally applied ZnCl2 reduced the H+ current, shifted the voltage-activation curve toward positive potentials, and slowed the turn-on of H+ current upon depolarization more than could be accounted for by a simple voltage shift, with minimal effects on the closing rate. The effects of Zn2+ were inconsistent with classical voltage-dependent block in which Zn2+ binds within the membrane voltage field. Instead, Zn2+ binds to superficial sites on the channel and modulates gating. The effects of extracellular Zn2+ were strongly pHo dependent but were insensitive to pHi, suggesting that protons and Zn2+ compete for external sites on H+ channels. The apparent potency of Zn2+ in slowing activation was ∼10× greater at pHo 7 than at pHo 6, and ∼100× greater at pHo 6 than at pHo 5. The pHo dependence suggests that Zn2+, not ZnOH+, is the active species. Evidently, the Zn2+ receptor is formed by multiple groups, protonation of any of which inhibits Zn2+ binding. The external receptor bound H+ and Zn2+ with pKa 6.2–6.6 and pKM 6.5, as described by several models. Zn2+ effects on the proton chord conductance–voltage (gH–V) relationship indicated higher affinities, pKa 7 and pKM 8. CdCl2 had similar effects as ZnCl2 and competed with H+, but had lower affinity. Zn2+ applied internally via the pipette solution or to inside-out patches had comparatively small effects, but at high concentrations reduced H+ currents and slowed channel closing. Thus, external and internal zinc-binding sites are different. The external Zn2+ receptor may be the same modulatory protonation site(s) at which pHo regulates H+ channel gating.
The Extracellular Linker of Muscle Acetylcholine Receptor Channels Is a Gating Control Element
Authors: Grosman Claudio , Salamone Frank N. , Sine Steven M. , Auerbach Anthony
Published: July 2002

We describe the functional consequences of mutations in the linker between the second and third transmembrane segments (M2–M3L) of muscle acetylcholine receptors at the single-channel level. Hydrophobic mutations (Ile, Cys, and Phe) placed near the middle of the linker of the α subunit (αS269) prolong apparent openings elicited by low concentrations of acetylcholine (ACh), whereas hydrophilic mutations (Asp, Lys, and Gln) are without effect. Because the gating kinetics of the αS269I receptor (a congenital myasthenic syndrome mutant) in the presence of ACh are too fast, choline was used as the agonist. This revealed an ∼92-fold increased gating equilibrium constant, which is consistent with an ∼10-fold decreased EC50 in the presence of ACh. With choline, this mutation accelerates channel opening ∼28-fold, slows channel closing ∼3-fold, but does not affect agonist binding to the closed state. These ratios suggest that, with ACh, αS269I acetylcholine receptors open at a rate of ∼1.4 × 106 s−1 and close at a rate of ∼760 s−1. These gating rate constants, together with the measured duration of apparent openings at low ACh concentrations, further suggest that ACh dissociates from the diliganded open receptor at a rate of ∼140 s−1. Ile mutations at positions flanking αS269 impair, rather than enhance, channel gating. Inserting or deleting one residue from this linker in the α subunit increased and decreased, respectively, the apparent open time approximately twofold. Contrary to the αS269I mutation, Ile mutations at equivalent positions of the β, ε, and δ subunits do not affect apparent open-channel lifetimes. However, in β and ε, shifting the mutation one residue to the NH2-terminal end enhances channel gating. The overall results indicate that this linker is a control element whose hydrophobicity determines channel gating in a position- and subunit-dependent manner. Characterization of the transition state of the gating reaction suggests that during channel opening the M2–M3L of the α subunit moves before the corresponding linkers of the β and ε subunits.
Light Adaptation in Drosophila Photoreceptors
Authors: Juusola Mikko , Hardie Roger C.
Published: July 2002

Besides the physical limits imposed on photon absorption, the coprocessing of visual information by the phototransduction cascade and photoreceptor membrane determines the fidelity of photoreceptor signaling. We investigated the response dynamics and signaling efficiency of Drosophila photoreceptors to natural-like fluctuating light contrast stimulation and intracellular current injection when the cells were adapted over a 4-log unit light intensity range at 25°C. This dual stimulation allowed us to characterize how an increase in the mean light intensity causes the phototransduction cascade and photoreceptor membrane to produce larger, faster and increasingly accurate voltage responses to a given contrast. Using signal and noise analysis, this appears to be associated with an increased summation of smaller and faster elementary responses (i.e., bumps), whose latency distribution stays relatively unchanged at different mean light intensity levels. As the phototransduction cascade increases, the size and speed of the signals (light current) at higher adapting backgrounds and, in conjunction with the photoreceptor membrane, reduces the light-induced voltage noise, and the photoreceptor signal-to-noise ratio improves and extends to a higher bandwidth. Because the voltage responses to light contrasts are much slower than those evoked by current injection, the photoreceptor membrane does not limit the speed of the phototransduction cascade, but it does filter the associated high frequency noise. The photoreceptor information capacity increases with light adaptation and starts to saturate at ∼200 bits/s as the speed of the chemical reactions inside a fixed number of transduction units, possibly microvilli, is approaching its maximum.
Atp-Dependent Adenophostin Activation of Inositol 1,4,5-Trisphosphate Receptor Channel Gating
Authors: Mak Don-On Daniel , McBride Sean , Foskett J. Kevin
Published: July 2002

The inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) is a ligand-gated intracellular Ca2+ release channel that plays a central role in modulating cytoplasmic free Ca2+ concentration ([Ca2+]i). The fungal metabolite adenophostin A (AdA) is a potent agonist of the InsP3R that is structurally different from InsP3 and elicits distinct calcium signals in cells. We have investigated the effects of AdA and its analogues on single-channel activities of the InsP3R in the outer membrane of isolated Xenopus laevis oocyte nuclei. InsP3R activated by either AdA or InsP3 have identical channel conductance properties. Furthermore, AdA, like InsP3, activates the channel by tuning Ca2+ inhibition of gating. However, gating of the AdA-liganded InsP3R has a critical dependence on cytoplasmic ATP free acid concentration not observed for InsP3-liganded channels. Channel gating activated by AdA is indistinguishable from that elicited by InsP3 in the presence of 0.5 mM ATP, although the functional affinity of the channel is 60-fold higher for AdA. However, in the absence of ATP, gating kinetics of AdA-liganded InsP3R were very different. Channel open time was reduced by 50%, resulting in substantially lower maximum open probability than channels activated by AdA in the presence of ATP, or by InsP3 in the presence or absence of ATP. Also, the higher functional affinity of InsP3R for AdA than for InsP3 is nearly abolished in the absence of ATP. Low affinity AdA analogues furanophostin and ribophostin activated InsP3R channels with gating properties similar to those of AdA. These results provide novel insights for interpretations of observed effects of AdA on calcium signaling, including the mechanisms that determine the durations of elementary Ca2+ release events in cells. Comparisons of single-channel gating kinetics of the InsP3R activated by InsP3, AdA, and its analogues also identify molecular elements in InsP3R ligands that contribute to binding and activation of channel gating.
Evidence That Both Ligand Binding and Covalent Adaptation Drive a Two-State Equilibrium in the Aspartate Receptor Signaling Complex
Authors: Bornhorst Joshua A. , Falke Joseph J.
Published: July 2002

The transmembrane aspartate receptor of bacterial chemotaxis regulates an associated kinase protein in response to both attractant binding to the receptor periplasmic domain and covalent modification of four adaptation sites on the receptor cytoplasmic domain. The existence of at least 16 covalent modification states raises the question of how many stable signaling conformations exist. In the simplest case, the receptor could have just two stable conformations (“on” and “off”) yielding the two-state behavior of a toggle-switch. Alternatively, covalent modification could incrementally shift the receptor between many more than two stable conformations, thereby allowing the receptor to function as a rheostatic switch. An important distinction between these models is that the observed functional parameters of a toggle-switch receptor could strongly covary as covalent modification shifts the equilibrium between the on- and off-states, due to population-weighted averaging of the intrinsic on- and off-state parameters. By contrast, covalent modification of a rheostatic receptor would create new conformational states with completely independent parameters. To resolve the toggle-switch and rheostat models, the present study has generated all 16 homogeneous covalent modification states of the receptor adaptation sites, and has compared their effects on the attractant affinity and kinase activity of the reconstituted receptor–kinase signaling complex. This approach reveals that receptor covalent modification modulates both attractant affinity and kinase activity up to 100-fold, respectively. The regulatory effects of individual adaptation sites are not perfectly additive, indicating synergistic interactions between sites. The three adaptation sites at positions 295, 302, and 309 are more important than the site at position 491 in regulating attractant affinity and kinase activity, thereby explaining the previously observed dominance of the former three sites in in vivo studies. The most notable finding is that covalent modification of the adaptation sites alters the receptor attractant affinity and the receptor-regulated kinase activity in a highly correlated fashion, strongly supporting the toggle-switch model. Similarly, certain mutations that drive the receptor into the kinase activating state are found to have correlated effects on attractant affinity. Together these results provide strong evidence that chemotaxis receptors possess just two stable signaling conformations and that the equilibrium between these pure on- and off-states is modulated by both attractant binding and covalent adaptation. It follows that the attractant and adaptation signals drive the same conformational change between the two settings of a toggle. An approach that quantifies the fractional occupancy of the on- and off-states is illustrated.
Separation and Characterization of Currents through Store-operated CRAC Channels and Mg2+-inhibited Cation (MIC) Channels
Authors: Prakriya Murali , Lewis Richard S.
Published: July 2002

Although store-operated calcium release–activated Ca2+(CRAC) channels are highly Ca2+-selective under physiological ionic conditions, removal of extracellular divalent cations makes them freely permeable to monovalent cations. Several past studies have concluded that under these conditions CRAC channels conduct Na+and Cs+with a unitary conductance of ∼40 pS, and that intracellular Mg2+modulates their activity and selectivity. These results have important implications for understanding ion permeation through CRAC channels and for screening potential CRAC channel genes. We find that the observed 40-pS channels are not CRAC channels, but are instead Mg2+-inhibited cation (MIC) channels that open as Mg2+is washed out of the cytosol. MIC channels differ from CRAC channels in several critical respects. Store depletion does not activate MIC channels, nor does store refilling deactivate them. Unlike CRAC channels, MIC channels are not blocked by SKF 96365, are not potentiated by low doses of 2-APB, and are less sensitive to block by high doses of the drug. By applying 8–10 mM intracellular Mg2+to inhibit MIC channels, we examined monovalent permeation through CRAC channels in isolation. A rapid switch from 20 mM Ca2+to divalent-free extracellular solution evokes Na+current through open CRAC channels (Na+-ICRAC) that is initially eightfold larger than the preceding Ca2+current and declines by ∼80% over 20 s. Unlike MIC channels, CRAC channels are largely impermeable to Cs+(PCs/PNa= 0.13 vs. 1.2 for MIC). Neither the decline in Na+-ICRACnor its low Cs+permeability are affected by intracellular Mg2+(90 μM to 10 mM). Single openings of monovalent CRAC channels were not detectable in whole-cell recordings, but a unitary conductance of 0.2 pS was estimated from noise analysis. This new information about the selectivity, conductance, and regulation of CRAC channels forces a revision of the biophysical fingerprint of CRAC channels, and reveals intriguing similarities and differences in permeation mechanisms of voltage-gated and store-operated Ca2+channels.
Vav Regulates Activation of Rac but Not Cdc42 during FcγR-mediated Phagocytosis
Authors: Patel Jayesh C. , Hall Alan , Caron Emmanuelle
Published: July 2002

Phagocytosis is the process whereby cells direct the spatially localized, receptor-driven engulfment of particulate materials. It proceeds via remodeling of the actin cytoskeleton and shares many of the core cytoskeletal components involved in adhesion and migration. Small GTPases of the Rho family have been widely implicated in coordinating actin dynamics in response to extracellular signals and during diverse cellular processes, including phagocytosis, yet the mechanisms controlling their recruitment and activation are not known. We show herein that in response to ligation of Fc receptors for IgG (FcγR), the guanine nucleotide exchange factor Vav translocates to nascent phagosomes and catalyzes GTP loading on Rac, but not Cdc42. The Vav-induced Rac activation proceeds independently of Cdc42 function, suggesting distinct roles for each GTPase during engulfment. Moreover, inhibition of Vav exchange activity or of Cdc42 activity does not prevent Rac recruitment to sites of particle attachment. We conclude that Rac is recruited to Fcγ membrane receptors in its inactive, GDP-bound state and that Vav regulates phagocytosis through subsequent catalysis of GDP/GTP exchange on Rac.
Alcohol Consumption and Bladder Cancer Risk: Results from the Netherlands Cohort Study
Authors: Zeegers Maurice P. A. , Volovics Alex , Dorant Elisabeth , Alexandra Goldbohm R. , van den Brandt Piet A.
Published: July 2002

Although several epidemiologic studies have been conducted on alcohol consumption and bladder cancer risk, the risk according to quantity and type of alcohol consumed is not clear. The authors investigated these associations in a large prospective cohort study on diet and cancer among 120,852 subjects in the Netherlands aged 55–69 years at baseline (1986). Subjects completed a questionnaire on risk factors for cancer, including alcohol consumption. Follow-up for incident cancer was established by record linkage to cancer registries. The case-cohort analysis was restricted to a follow-up period of 6.3 years and was based on 594 cases with bladder cancer and 3,170 subcohort members. The authors corrected for age and smoking in multivariable analyses. The incidence rate ratios for men who consumed <5, 5–<15, 15–<30, and ≥30 grams of alcohol per day were 1.49, 1.52, 1.16, and 1.63 compared with nondrinkers, respectively (p for trend = 0.13). Alcohol consumed from beer, wine, and liquor was associated with moderately elevated risks, although most were not statistically significant. The incidence rate ratios for women varied around unity. The results of this study do not suggest an important association between alcohol consumption and bladder cancer risk.
Case-Control Study on Lung Cancer and Residential Radon in Western Germany
Authors: Kreienbrock Lothar , Kreuzer Michaela , Gerken Michael , Dingerkus Gerlinde , Wellmann Jürgen , Keller Gert , Erich Wichmann H.
Published: July 2002

In a 1990–1996 case-control study in western Germany, the authors investigated lung cancer risk due to exposure to residential radon. Confirmed lung cancer cases from hospitals and a random sample of community controls were interviewed by trained interviewers regarding different risk factors. For 1 year, alpha track detectors were placed in dwellings to measure radon gas concentrations. The evaluation included 1,449 cases and 2,297 controls recruited from the entire study area and a subsample of 365 cases and 595 controls from radon-prone areas of the basic study region. Rate ratios were estimated by using conditional logistic regression adjusted for smoking and for asbestos exposure. In the entire study area, no rate ratios different from 1.0 were found; in the radon-prone areas, the adjusted rate ratios for exposure in the present dwelling were 1.59 (95% confidence interval (CI): 1.08, 2.27), 1.93 (95% CI: 1.19, 3.13), and 1.93 (95% CI: 0.99, 3.77) for 50–80, 80–140, and >140 Bq/m3, respectively, compared with 0–50 Bq/m3. The excess rate ratio for an increase of 100 Bq/m3 was 0.13 (−0.12 to 0.46). An analysis based on cumulative exposure produced similar results. The results provide additional evidence that residential radon is a risk factor for lung cancer, although a risk was detected in radon-prone areas only, not in the entire study area.
Impact of Estrogen Use on Decline in Cognitive Function in a Representative Sample of Older Community-resident Women
Authors: Fillenbaum Gerda G. , Hanlon Joseph T. , Landerman Lawrence R. , Schmader Kenneth E.
Published: July 2002

The authors investigated whether postmenopausal estrogen use helps to maintain cognitive function; a brief screen, the Short Portable Mental Status Questionnaire (SPMSQ), was used. Information was gathered from a stratified, random sample of 1,907 African-American and White women (aged 65–100 years) participating in the longitudinal Duke Established Populations for Epidemiologic Studies of the Elderly project carried out in five urban and rural counties of North Carolina. All women were cognitively unimpaired in 1986–1987 and were evaluated 3 and 6 years later. Decline in cognitive function was measured as an increase of two or more errors on the SPMSQ and crossing of an SPMSQ threshold indicative of cognitive impairment. Recency and continuity of estrogen use were measured. Univariate analyses indicated that recent (crude odds ratio = 0.42, 95% confidence interval: 0.21, 0.86) and continuous (crude odds ratio = 0.32, 95% confidence interval: 0.13, 0.81) estrogen use reduced the risk of cognitive decline but not of cognitive impairment. After adjustment for demographic and health characteristics, protective effects became nonsignificant. While postmenopausal use of estrogen may be protective for Alzheimer's disease, current findings based on a brief cognitive screen suggest that it is not protective for cognitive decline related to aging.
Observed versus Indirect Estimates of Incidence of Open-Angle Glaucoma
Authors: Wu Suh-Yuh , Nemesure Barbara , Leske M. Cristina
Published: July 2002

Incidence data on open-angle glaucoma (OAG) are limited and difficult to obtain. To date, few studies have reported incidence directly measured from population-based cohorts. Other reported estimates have been derived indirectly from age-specific prevalence by using several assumptions, and their validity is unknown. To the authors' knowledge, this report presents the first comparison of observed versus indirect estimates of OAG incidence based on data from the population-based Barbados Incidence Study of Eye Diseases (1992–1997) (n = 3,427; 85% participation). The observed 4-year incidence of OAG was 1.2% (95% confidence interval (CI): 0.6, 2.1%) at ages 40–49 years, 1.5% (95% CI: 0.8, 2.5%) at ages 50–59 years, 3.2% (95% CI: 2.0, 4.8%) at ages 60–69 years, and 4.2% (95% CI: 2.6, 6.3%) in persons at ages 70 or more years. When incidence was calculated from the prevalence data, power function fitting achieved a closer approximation to observed incidence than did logistic curve fitting. Calculated incidence rates for each group were similar when assuming mortality that was equal (incidence rate = 0.7, 1.3, 2.3, and 4.8%) or differential (incidence rate = 0.7, 1.2, 2.4, and 4.8%). Other nonlogistic approaches also increased the resemblance of observed and calculated estimates. In the absence of longitudinal data, reasonably valid incidence estimates of OAG were obtained from available prevalence data. These estimation techniques can be useful when OAG incidence estimates are required for research or public health purposes.
Familial Risk Ratio of Sarcoidosis in African-American Sibs and Parents
Authors: Rybicki Benjamin A. , Kirkey Kandace L. , Major Marcie , Maliarik Mary J. , Popovich John , Chase Gary A. , Iannuzzi Michael C.
Published: July 2002

While sarcoidosis is thought to aggregate in families, little is known about the risk to relatives of sarcoidosis patients. To estimate the familial risk ratio (λ) of sarcoidosis in sibs and parents of cases, the authors studied 179 African-American families ascertained through an index sarcoidosis case diagnosed at Henry Ford Hospital in Detroit, Michigan. Among those relatives enrolled between 1997 and 1999, 12 of 327 (3.7%) sibs and 11 of 161 (6.8%) parents reported a history of sarcoidosis. The λ in this sample of relatives, estimated by computing an age, sex, and race standardized incidence ratio, was 2.24 (95% confidence interval: 1.16, 3.92) for sibs and 2.82 (95% confidence interval: 1.41, 5.05) for parents. For sibs and parents combined, λ was 2.49 (95% confidence interval: 1.58, 3.73). Results stratified by proband characteristics indicated that λ was greater for relatives of younger (λ = 2.93, 95% confidence interval: 1.52, 5.12) and male (λ = 3.98, 95% confidence interval: 1.99, 7.12) probands. A higher λ was also found for male family members and sibs born later in the birth order. A Monte Carlo method was also used to estimate λ, with similar results obtained. Overall, these results indicate that, in African Americans, sibs and parents of sarcoidosis cases have about a 2.5-fold increased risk for sarcoidosis and that heterogeneity in disease risk may exist among family members.