bluuebunny/crossref_metadata_2025 huggingface dataset:
The Level of Virus-Specific T-Cell and Macrophage Recruitment in Porcine Reproductive and Respiratory Syndrome Virus Infection in Pigs Is Independent of Virus Load
Authors: Xiao Zhengguo , Batista Laura , Dee Scott , Halbur Patrick , Murtaugh Michael P.
Published: May 2004

Porcine reproductive and respiratory syndrome virus (PRRSV) is the most important infectious disease agent of pigs worldwide, causing reproductive failure in pregnant sows and respiratory problems in nursing and growing pigs. PRRSV infection is characterized by a prolonged viremia of 30 or more days and an extended persistent infection of lymphoid tissues. To better understand the immunological basis for prolonged acute and persistent PRRSV infection, we have examined the cell-mediated immune (CMI) response throughout the course of infection and compared the results to the local distribution and abundance of PRRSV in infected tissues. PRRSV-specific T cells, enumerated by gamma interferon enzyme-linked immunospot assay, did not appear until 2 weeks after PRRSV inoculation, and their abundance exhibited substantial variation over time and among animals. In all cases the T-cell response was transient. High levels of viral RNA were present in lymphoid tissues of all animals in the acute phase of infection. Viral loads were decreased 1,000-fold or more in persistent infections, with the primary sites of persistence being tonsil, sternal lymph node, and inguinal lymph node. The abundance of virus-specific T cells in either acutely or persistently infected animals was highly variable and showed no correlation to the level of virus in lymphoid tissues. No significant difference in antigen-specific T-cell abundance was observed in secondary lymphoid tissues in either acute or persistent infection except for tonsil, in which the number of responding cells was extremely low. CD4+- and CD8+-T-cell frequencies did not change after PRRSV infection, though a decrease in γδ T cells was observed. Macrophages, the permissive cell type for PRRSV, were present in various levels in all tissue preparations and were not in proportion to local virus load. These findings indicate that a weak CMI response contributes to prolonged PRRSV infection and suggests that PRRSV suppresses T-cell recognition of infected macrophages. Thus, the slow but eventual resolution of PRRSV infection may be dependent on limiting permissive macrophages and on innate immune factors.
Mutational Analyses of Epstein-Barr Virus Glycoprotein 42 Reveal Functional Domains Not Involved in Receptor Binding but Required for Membrane Fusion
Authors: Silva Amanda L. , Omerović Jasmina , Jardetzky Theodore S. , Longnecker Richard
Published: May 2004

Epstein-Barr virus (EBV) is a human gammaherpesvirus associated with malignancies of both epithelial and lymphoid origin. Efficient infection of the latent host reservoir B lymphocytes involves the binding of glycoproteins gp350/220 for initial attachment, followed by the concerted action of gH, gL, gB, and gp42 for membrane fusion. The type II membrane protein gp42 is required for infection of B cells and assembles into a complex with gH and gL. The cellular host receptor for gp42, class II human leukocyte antigen (HLA), has been structurally verified by crystallization analyses of gp42 bound to HLA-DR1. Interestingly, the crystal structure revealed a hydrophobic pocket consisting of many aromatic and aliphatic residues from the predicted C-type lectin domain of gp42 that in other members of the C-type lectin family binds major histocompatibility complex class I or other diverse ligands. Although the hydrophobic pocket does not bind HLA class II, mutational analyses presented here indicate that this domain is essential for EBV-induced membrane fusion. In addition, mutational analysis of the region of gp42 contacting HLA class II in the gp42-HLA-DR1 cocrystal confirms that this region interacts with HLA class II and that this interaction is also important for EBV-induced membrane fusion.
Cleavage between Replicase Proteins p28 and p65 of Mouse Hepatitis Virus Is Not Required for Virus Replication
Authors: Denison Mark R. , Yount Boyd , Brockway Sarah M. , Graham Rachel L. , Sims Amy C. , Lu XiaoTao , Baric Ralph S.
Published: May 2004

The p28 and p65 proteins of mouse hepatitis virus (MHV) are the most amino-terminal protein domains of the replicase polyprotein. Cleavage between p28 and p65 has been shown to occur in vitro at cleavage site 1 (CS1),
247
Gly↓Val
248
, in the polyprotein. Although critical residues for CS1 cleavage have been mapped in vitro, the requirements for cleavage have not been studied in infected cells. To define the determinants of CS1 cleavage and the role of processing at this site during MHV replication, mutations and deletions were engineered in the replicase polyprotein at CS1. Mutations predicted to allow cleavage at CS1 yielded viable virus that grew to wild-type MHV titers and showed normal expression and processing of p28 and p65. Mutant viruses containing predicted noncleaving mutations or a CS1 deletion were also viable but demonstrated delayed growth kinetics, reduced peak titers, decreased RNA synthesis, and small plaques compared to wild-type controls. No p28 or p65 was detected in cells infected with predicted noncleaving CS1 mutants or the CS1 deletion mutant; however, a new protein of 93 kDa was detected. All introduced mutations and the deletion were retained during repeated virus passages in culture, and no phenotypic reversion was observed. The results of this study demonstrate that cleavage between p28 and p65 at CS1 is not required for MHV replication. However, proteolytic separation of p28 from p65 is necessary for optimal RNA synthesis and virus growth, suggesting important roles for these proteins in the formation or function of viral replication complexes.
Intracellular Topology and Epitope Shielding of Poliovirus 3A Protein
Authors: Choe Sunny S. , Kirkegaard Karla
Published: May 2004

The poliovirus RNA replication complex comprises multiple viral and possibly cellular proteins assembled on the cytoplasmic surface of rearranged intracellular membranes. Viral proteins 3A and 3AB perform several functions during the poliovirus replicative cycle, including significant roles in rearranging membranes, anchoring the viral polymerase to these membranes, inhibiting host protein secretion, and possibly providing the 3B protein primer for RNA synthesis. During poliovirus infection, the immunofluorescence signal of an amino-terminal epitope of 3A-containing proteins is markedly shielded compared to 3A protein expressed in the absence of other poliovirus proteins. This is not due to luminal orientation of all or a subset of the 3A-containing polypeptides, as shown by immunofluorescence following differential permeabilization and proteolysis experiments. Shielding of the 3A epitope is more pronounced in cells infected with wild-type poliovirus than in cells with temperature-sensitive mutant virus that contains a mutation in the 3D polymerase coding region adjacent to the 3AB binding site. Therefore, it is likely that direct binding of the poliovirus RNA-dependent RNA polymerase occludes the amino terminus of 3A-containing polypeptides in the RNA replication complex.
Differential Effects of Interleukin-7 and Interleukin-15 on NK Cell Anti-Human Immunodeficiency Virus Activity
Authors: Lum Julian J. , Schnepple David J. , Nie Zilin , Sanchez-Dardon Jaime , Mbisa Georgina L. , Mihowich Jennifer , Hawley Nanci , Narayan Shanil , Kim John E. , Lynch David H. , Badley Andrew D.
Published: May 2004

The ability of interleukin-7 (IL-7) and IL-15 to expand and/or augment effector cell functions may be of therapeutic benefit to human immunodeficiency virus (HIV)-infected patients. The functional effects of these cytokines on innate HIV-specific immunity and their impact on cells harboring HIV are unknown. We demonstrate that both IL-7 and IL-15 augment natural killer (NK) function by using cells (CD3−CD16+CD56+) from both HIV-positive and -negative donors. Whereas IL-7 enhances NK function through upregulation of Fas ligand, the effect of IL-15 is mediated through upregulation of tumor necrosis factor-related apoptosis-inducing ligand. The difference in these effector mechanisms is reflected by the ability of IL-15-treated but not IL-7-treated NK cells to reduce the burden of replication-competent HIV in autologous peripheral blood mononuclear cells (PBMC) (infectious units per million for control NK cells, 6.79; for IL-7-treated NK cells, 236.17; for IL-15-treated cells, 1.01;P= 0.01 versus control). In addition, the treatment of PBMC with IL-15-treated but not IL-7-treated NK cells causes undetectable HIV p24 (five of five cases), HIV RNA (five of five cases), or HIV DNA (three of five cases). These results support the concept of adjuvant immunotherapy of HIV infection with either IL-7 or IL-15 but suggest that the NK-mediated antiviral effect of IL-15 may be superior.
Reverse Genetic Analysis of the Transcription Regulatory Sequence of the Coronavirus Transmissible Gastroenteritis Virus
Authors: Curtis Kristopher M. , Yount Boyd , Sims Amy C. , Baric Ralph S.
Published: May 2004

Coronavirus discontinuous transcription uses a highly conserved sequence (CS) in the joining of leader and body RNAs. Using a full-length infectious construct of transmissable gastroenteritis virus, the present study demonstrates that subgenomic transcription is heavily influenced by upstream flanking sequences and supports a mechanism of transcription attenuation that is regulated in part by a larger domain composed of primarily upstream flanking sequences which select appropriately positioned CS elements for synthesis of subgenomic RNAs.
HETEROCLINIC CONTOURS IN NEURAL ENSEMBLES AND THE WINNERLESS COMPETITION PRINCIPLE
Authors: AFRAIMOVICH VALENTIN S. , RABINOVICH MIKHAIL I. , VARONA PABLO
Published: May 2004

The ability of nonlinear dynamical systems to process incoming information is a key problem of many fundamental and applied sciences. Information processing by computation with attractors (steady states, limit cycles and strange attractors) has been a subject of many publications. In this paper, we discuss a new direction in information dynamics based on neurophysiological experiments that can be applied for the explanation and prediction of many phenomena in living biological systems and for the design of new paradigms in neural computation. This new concept is the Winnerless Competition (WLC) principle. The main point of this principle is the transformation of the incoming identity or spatial inputs into identity-temporal output based on the intrinsic switching dynamics of the neural system. In the presence of stimuli the sequence of the switching, whose geometrical image in the phase space is a heteroclinic contour, uniquely depends on the incoming information. The key problem in the realization of the WLC principle is the robustness against noise and, simultaneously, the sensitivity of the switching to the incoming input. In this paper we prove two theorems about the stability of the sequential switching and give several examples of WLC networks that illustrate the coexistence of sensitivity and robustness.
BIFURCATION AND CHAOS IN COUPLED BVP OSCILLATORS
Authors: UETA TETSUSHI , MIYAZAKI HISAYO , KOUSAKA TAKUJI , KAWAKAMI HIROSHI
Published: May 2004

Bonhöffer–van der Pol(BVP) oscillator is a classic model exhibiting typical nonlinear phenomena in the planar autonomous system. This paper gives an analysis of equilibria, periodic solutions, strange attractors of two BVP oscillators coupled by a resister. When an oscillator is fixed its parameter values in nonoscillatory region and the others in oscillatory region, create the double scroll attractor due to the coupling. Bifurcation diagrams are obtained numerically from the mathematical model and chaotic parameter regions are clarified. We also confirm the existence of period-doubling cascades and chaotic attractors in the experimental laboratory.
Conformational Spread: The Propagation of Allosteric States in Large Multiprotein Complexes
Authors: Bray Dennis , Duke Thomas
Published: May 2004

▪ Abstract  The phenomenon of allostery is conventionally described for small symmetrical oligomeric proteins such as hemoglobin. Here we review experimental evidence from a variety of systems—including bacterial chemotaxis receptors, muscle ryanodine receptors, and actin filaments—showing that conformational changes can also propagate through extended lattices of protein molecules. We explore the statistical mechanics of idealized linear and two-dimensional arrays of allosteric proteins and show that, as in the analogous Ising models, arrays of closely packed units can show large-scale integrated behavior. We also discuss proteins that undergo conformational changes driven by the hydrolysis of ATP and give examples in which these changes propagate through linear chains of molecules. We suggest that conformational spread could provide the basis of a solid-state “circuitry” in a living cell, able to integrate biochemical and biophysical events over hundreds of protein molecules.
The Use of In Vitro Peptide-Library Screens in the Analysis of Phosphoserine/Threonine-Binding Domain Structure and Function
Authors: Yaffe Michael B. , Smerdon Stephen J.
Published: May 2004

▪ Abstract  Phosphoserine/threonine-binding domains integrate intracellular signal transduction events by forming multiprotein complexes with substrates of protein serine/threonine kinases. These phosphorylation-dependent molecular recognition events are responsible for coordinating the precise temporal and spatial response of cells to a wide range of stimuli, particularly those involved in cell cycle control and the response to DNA damage. The known families of phosphoserine/threonine-binding modules include 14-3-3 proteins, WW domains, FHA domains, WD40 repeats, and the Polo-box domains of Polo-like kinases. Peptide-library experiments reveal the optimal sequence motifs recognized by these domains, and facilitate high-resolution structural studies elucidating the mechanisms of phospho-dependent binding and the molecular basis for domain function within intricate signaling networks. Information emerging from these studies is critical for the design of novel experimental and therapeutic tools aimed at altering signal transduction cascades in normal and diseased cells.
Spin Distribution and the Location of Protons in Paramagnetic Proteins
Authors: Goldfarb D. , Arieli D.
Published: May 2004

▪ Abstract  Two current frontiers in EPR research are high-field ([Formula: see text]) electron paramagnetic resonance (EPR) and high-field electron-nuclear double resonance (ENDOR). This review focuses on recent advances in high-field ENDOR and its applications to the study of proteins containing native paramagnetic sites. It concentrates on two aspects; the first concerns the determination of the location of protons and is related to the site geometry, and the second focuses on the spin density distribution within the site, which is inherent to the electronic structure. Both spin density and proton locations can be derived from ligand hyperfine couplings determined by ENDOR measurements. A brief description of the experimental methods is presented along with a discussion of the advantages and disadvantages of high-field ENDOR compared with conventional X-band (∼ 9.5 GHz) experiments. Specific examples of both protein single crystals and frozen solutions are then presented. These include the determination of the coordinates of water ligand protons in the Mn(II) site of concanavalin A, the detection of hydrogen bonds in a quinone radical in the bacterial photosynthetic reaction center as well as in the tyrosyl radical in ribonuclease reductase, and the study of the spin distribution in copper proteins. The copper proteins discussed are the type I copper of azurin and the binuclear CuAcenter in a number of proteins. The last part of the review presents a brief discussion of the interpretation of hyperfine couplings using quantum chemical calculations, primarily density functional theory (DFT) methods. Such methods are becoming an integral part of the data analysis tools, as they can facilitate signal assignment and provide the ultimate relation between the experimental hyperfine couplings and the electronic wave function.
Structure, Molecular Mechanisms, and Evolutionary Relationships in DNA Topoisomerases
Authors: Corbett Kevin D. , Berger James M.
Published: May 2004

▪ Abstract  Topoisomerases are enzymes that use DNA strand scission, manipulation, and rejoining activities to directly modulate DNA topology. These actions provide a powerful means to effect changes in DNA supercoiling levels, and allow some topoisomerases to both unknot and decatenate chromosomes. Since their initial discovery over three decades ago, researchers have amassed a rich store of information on the cellular roles and regulation of topoisomerases, and have delineated general models for their chemical and physical mechanisms. Topoisomerases are now known to be necessary for the survival of cellular organisms and many viruses and are rich clinical targets for anticancer and antimicrobial treatments. In recent years, crystal structures have been obtained for each of the four types of topoisomerases in a number of distinct conformational and substrate-bound states. In addition, sophisticated biophysical methods have been utilized to study details of topoisomerase reaction dynamics and enzymology. A synthesis of these approaches has provided researchers with new physical insights into how topoisomerases employ chemistry and allostery to direct the large-scale molecular motions needed to pass DNA strands through each other.
Taking X-Ray Diffraction to the Limit: Macromolecular Structures from Femtosecond X-Ray Pulses and Diffraction Microscopy of Cells with Synchrotron Radiation
Authors: Miao Jianwei , Chapman Henry N. , Kirz Janos , Sayre David , Hodgson Keith O.
Published: May 2004

▪ Abstract  Recent work is extending the methodology of X-ray crystallography to the structure determination of noncrystalline specimens. The phase problem is solved using the oversampling method, which takes advantage of “continuous” diffraction patterns from noncrystalline specimens. Here we review the principle of this newly developed technique and discuss the ongoing experiments of imaging nonperiodic objects, such as cells and cellular structures, using coherent and bright X rays produced by third-generation synchrotron sources. In the longer run, the technique may be applicable to image single biomolecules using anticipated X-ray free electron lasers. Here, computer simulations have so far demonstrated two important steps: (a) by using an extremely intense femtosecond X-ray pulse, a diffraction pattern can be recorded from a macromolecule before radiation damage manifests itself; and (b) the phase information can be retrieved in an ab initio fashion from a set of calculated noisy diffraction patterns of single protein molecules.
Tethering: Fragment-Based Drug Discovery
Authors: Erlanson Daniel A. , Wells James A. , Braisted Andrew C.
Published: May 2004

▪ Abstract  The genomics revolution has provided a deluge of new targets for drug discovery. To facilitate the drug discovery process, many researchers are turning to fragment-based approaches to find lead molecules more efficiently. One such method, Tethering 1 , allows for the identification of small-molecule fragments that bind to specific regions of a protein target. These fragments can then be elaborated, combined with other molecules, or combined with one another to provide high-affinity drug leads. In this review we describe the background and theory behind Tethering and discuss its use in identifying novel inhibitors for protein targets including interleukin-2 (IL-2), thymidylate synthase (TS), protein tyrosine phosphatase 1B (PTP-1B), and caspases.
The Role of Water in Protein-DNA Recognition
Authors: Jayaram B. , Jain Tarun
Published: May 2004

▪ Abstract  Is it by design or by default that water molecules are observed at the interfaces of some protein-DNA complexes? Both experimental and theoretical studies on the thermodynamics of protein-DNA binding overwhelmingly support the extended hydrophobic view that water release from interfaces favors binding. Structural and energy analyses indicate that the waters that remain at the interfaces of protein-DNA complexes ensure liquid-state packing densities, screen the electrostatic repulsions between like charges (which seems to be by design), and in a few cases act as linkers between complementary charges on the biomolecules (which may well be by default). This review presents a survey of the current literature on water in protein-DNA complexes and a critique of various interpretations of the data in the context of the role of water in protein-DNA binding and principles of protein-DNA recognition in general.
Glucagon-like peptide 1: evolution of an incretin into a treatment for diabetes
Authors: D'Alessio David A. , Vahl Torsten P.
Published: May 2004

Glucagon-like peptide 1 (GLP-1) is a product of proglucagon that is secreted by specialized intestinal endocrine cells after meals. GLP-1 is insulinotropic and plays a role in the incretin effect, the augmented insulin response observed when glucose is absorbed through the gut. GLP-1 also appears to regulate a number of processes that reduce fluctuations in blood glucose, such as gastric emptying, glucagon secretion, food intake, and possibly glucose production and glucose uptake. These effects, in addition to the stimulation of insulin secretion, suggest a broad role for GLP-1 as a mediator of postprandial glucose homeostasis. Consistent with this role, the most prominent effect of experimental blockade of GLP-1 signaling is an increase in blood glucose. Recent data also suggest that GLP-1 is involved in the regulation of β-cell mass. Whereas other insulinotropic gastrointestinal hormones are relatively ineffective in stimulating insulin secretion in persons with type 2 diabetes, GLP-1 retains this action and is very effective in lowering blood glucose levels in these patients. There are currently a number of products in development that utilize the GLP-1-signaling system as a mechanism for the treatment of diabetes. These compounds, GLP-1 receptor agonists and agents that retard the metabolism of native GLP-1, have shown promising results in clinical trials. The application of GLP-1 to clinical use fulfills a long-standing interest in adapting endogenous insulinotropic hormones to the treatment of diabetes.
Insulin action during late pregnancy in the conscious dog
Authors: Connolly Cynthia C. , Aglione Lisa N. , Smith Marta S. , Lacy D. Brooks , Moore Mary Courtney
Published: May 2004

Our aim was to assess the magnitude of peripheral insulin resistance and whether changes in hepatic insulin action were evident in a canine model of late (3rd trimester) pregnancy. A 3-h hyperinsulinemic (5 mU·kg−1·min−1) euglycemic clamp was conducted using conscious, 18-h-fasted pregnant (P; n = 6) and nonpregnant (NP; n = 6) female dogs in which catheters for intraportal insulin infusion and assessment of hepatic substrate balances were implanted ∼17 days before experimentation. Arterial plasma insulin rose from 11 ± 2 to 192 ± 24 and 4 ± 2 to 178 ± 5 μU/ml in the 3rd h in NP and P, respectively. Glucagon fell equivalently in both groups. Basal net hepatic glucose output was lower in NP (1.9 ± 0.1 vs. 2.4 ± 0.2 mg·kg−1·min−1, P < 0.05). Hyperinsulinemia completely suppressed hepatic glucose release in both groups (−0.4 ± 0.2 and −0.1 ± 0.2 mg·kg−1·min−1 in NP and P, respectively). More exogenous glucose was required to maintain euglycemia in NP (15.2 ± 1.3 vs. 11.5 ± 1.1 mg·kg−1·min−1, P < 0.05). Nonesterified fatty acids fell similarly in both groups. Net hepatic gluconeogenic amino acid uptake with high insulin did not differ in NP and P. Peripheral insulin action is markedly impaired in this canine model of pregnancy, whereas hepatic glucose production is completely suppressed by high circulating insulin levels.
Distinct pathways regulate facilitated glucose transport in human articular chondrocytes during anabolic and catabolic responses
Authors: Shikhman Alexander R. , Brinson Diana C. , Lotz Martin K.
Published: May 2004

Articular cartilage is an avascular, non-insulin-sensitive tissue that utilizes glucose as the main energy source, a precursor for glycosaminoglycan synthesis, and a regulator of gene expression. Facilitated glucose transport represents the first rate-limiting step in glucose metabolism. Previously, we demonstrated that glucose transport in chondrocytes is regulated by proinflammatory cytokines via upregulation of GLUT mRNA and protein expression. The objective of the present study was to determine differences in molecular mechanisms regulating glucose transport in chondrocytes stimulated with the anabolic transforming growth factor-β1 (TGF-β1) vs. the catabolic and proinflammatory cytokine IL-1β. Both TGF-β1 and IL-1β accelerate glucose transport in chondrocytes. Although both IL-1β and TGF-β1 enhance glucose transport in chondrocytes to a similar magnitude, IL-1β induces significantly higher levels of lactate. TGF-β1-stimulated glucose transport is not associated with increased expression or membrane incorporation of GLUT1, -3, -6, -8, and -10 and depends on PKC and ERK activation. In contrast, IL-1β-stimulated glucose transport is accompanied by increased expression and membrane incorporation of GLUT1 and -6 and depends upon activation of PKC and p38 MAP kinase. In conclusion, anabolic and catabolic stimuli regulate facilitated glucose transport in human articular chondrocytes via different effector and signaling mechanisms, and they have distinct effects on glycolysis.
Parathyroid responsiveness to hypocalcemic and hypercalcemic stimuli in adult growth hormone deficiency after growth hormone replacement
Authors: Ahmad Aftab M. , Hopkins Marion T. , Thomas Joegi , Durham Brian H. , Fraser William D. , Vora Jiten P.
Published: May 2004

Adult growth hormone deficiency (AGHD) is associated with osteoporosis. Previous reports have suggested that alterations in parathyroid gland responsiveness to changes in calcium concentration may play a role in the genesis of osteoporosis in untreated AGHD patients. We investigated the endogenous parathyroid hormone [PTH-(1–84)] response to hypocalcemic and hypercalcemic stimuli induced by sodium EDTA and calcium gluconate infusion, respectively, and to PTH-(1–34) infusion in AGHD patients before and during GH replacement (GHR). We have demonstrated that the maximum PTH-(1–84) stimulation and suppression occurred at significantly higher calcium concentrations and in response to smaller changes in calcium concentrations after GHR. The calcemic response to the effects of PTH-(1–34) infusion significantly increased after GHR. The calcium set point (the calcium concentration at which the rate of PTH secretion is one-half of its maximal value) significantly increased in all groups after 3 mo on GHR, and it increased further at 12 mo. Our results suggest increased parathyroid gland sensitivity to smaller changes in serum calcium and increased end-organ sensitivity to the effects of PTH in AGHD patients after GHR. These findings may help us to understand the mechanisms underlying the genesis of osteoporosis in AGHD patients.
Insulin-like growth factor I receptors are more abundant than insulin receptors in human micro- and macrovascular endothelial cells
Authors: Chisalita Simona I. , Arnqvist Hans J.
Published: May 2004

Micro- and macroangiopathy are major causes of morbidity and mortality in patients with diabetes. Our aim was to characterize IGF-I receptor (IGF-IR) and insulin receptor (IR) in human micro- and macrovascular endothelial cells. Cultured human dermal microvascular endothelial cells (HMVEC) and human aortic endothelial cells (HAEC) were used. Gene expression was measured by quantitative real-time RT-PCR and receptor protein by ligand-binding assay. Phosphorylation of IGF-IR β-subunit was analyzed by immunoprecipitation and Western blot. Glucose metabolism and DNA synthesis was assessed using [3H]glucose and [3H]thymidine incorporation, respectively. We detected gene expression of IGF-IR and IR in HAEC and HMVEC. IGF-IR gene expression was severalfold higher than that of IR. The specific binding of125I-IGF-I was higher than that of125I-insulin in HAEC and HMVEC. Insulin and the new, long-acting insulin analog glargine interacted with the IGF-IR with thousand- and hundred-fold less potency than IGF-I itself. Phosphorylation of the IGF-IR β-subunit was shown in HAEC for IGF-I (10−8M) and insulin (10−6M) and in HMVEC for IGF-I and glargine (10−8M, 10−6M). IGF-I 10−7M stimulated incorporation of [3H]thymidine into DNA, and 10−9–10−7M also the incorporation of [3H]glucose in HMVEC, whereas glargine and insulin had no significant effects at 10−9–10−7M. Human micro- and macrovascular endothelial cells express more IGF-IR than IR. IGF-I and high concentrations of glargine and insulin activates the IGF-IR. Glargine has a higher affinity than insulin for the IGF-IR but probably has no effect on DNA synthesis at concentrations reached in vivo.
Characterization of the portal signal during 24-h glucose delivery in unrestrained, conscious rats
Authors: Ogihara N. , Kawamura W. , Kasuga K. , Hayashi Y. , Arakawa H. , Kikuchi M.
Published: May 2004

To characterize the “portal signal” during physiological glucose delivery, liver glycogen was measured in unrestrained rats during portal (Po) and peripheral (Pe) constant-rate infusion, with minimal differences in hepatic glucose load (HGL) and portal insulin between the delivery routes. Hepatic blood flows were measured by Doppler flowmetry during open surgery. Changes in hepatic glucose, portal insulin, glucagon, lactate, and free fatty acid concentrations were generally similar in either delivery except for glucagon at 4 h. Hepatic glycogen, however, increased continuously in Po and was higher than Pe at 8 and 24 h, although it decreased to the level of Pe upon the removal of Po at 8 h. There was a near-linear relationship between hepatic glycogen and HGL in either delivery, with the slope being twice as high in Po and the intercepts converging to basal HGL. The hepatic response to Po did not alter upon 80% replacement by Pe. These results suggest that negative arterial-portal glucose gradients increase the rate of hepatic glycogen synthesis against the incremental HGL in an all-or-nothing mode.
Protein kinase Cι enhances the transcriptional activity of the porcineP-450 side-chain cleavage insulin-like response element
Authors: Urban Randall J. , Bodenburg Yvonne H. , Jiang Jie , Denner Larry , Chedrese Jorge
Published: May 2004

IGF-I enhances steroidogenesis in granulosa cells by stimulating the expression of the rate-limiting steroidogenic enzyme, cytochrome P-450 side-chain cleavage ( P-450scc). This effect is mediated through an IGF response element (IGFRE) that binds polypyrimidine tract-binding protein (PTB)-associated splicing factor (PSF) and Sp1. Sp1 is essential for activation of the IGFRE, and PSF functions as a repressor. We investigated mechanisms of modulation of the IGFRE by the atypical protein kinase C (PKC)ι in a porcine stable granulosa cell line, JC-410. PKCι was found in nuclear extracts, and levels were increased by IGF-I after 24 and 48 h of treatment. Immunoprecipitation experiments demonstrated that PSF and PKCι associated with each other in nuclear extracts from JC-410 cells. Transient transfection with expression plasmids of kinase-active and kinase-deficient PKCι isoforms enhanced transcriptional activity of the IGFRE regardless of kinase catalytic activity. Depletion of PKCι protein by small interfering RNA suppressed basal IGFRE activity but did not prevent IGF-I stimulation of the IGFRE. We conclude that PKCι enhances transcriptional activity of the porcine P-450sccIGFRE independently of kinase activity by a mechanism involving protein-protein interaction with PSF.
Adipose depot-specific modulation of angiotensinogen gene expression in diet-induced obesity
Authors: Rahmouni Kamal , Mark Allyn L. , Haynes William G. , Sigmund Curt D.
Published: May 2004

Adipose tissue represents an important source of angiotensinogen (AGT). We investigated the effect of obesity induced by a high-fat diet on the expression of mouse (mAGT) and human AGT (hAGT) genes in liver, kidney, and heart and different adipose depots in normal mice (C57BL/6J), and in transgenic mice expressing the hAGT gene under the control of its own promoter. Mice were fed a high-fat diet (45% kcal) or normal chow (10% kcal) for 10 and 20 wk. The expression of mAGT and hAGT mRNA was quantified using an RNAse protection assay. Mice on the high-fat diet exhibited increased weight, fat mass, and plasma leptin. Expression of mAGT or hAGT genes was not affected by high-fat diet in nonadipose tissues, brown adipose tissue, or subcutaneous white fat. In contrast, high-fat diet increased both mAGT and hAGT gene expression in visceral adipose depots (omental, reproductive, and perirenal fat). Thus obesity-induced by a high-fat diet is associated with a tissue-specific increased expression of both mouse and human AGT genes in intra-abdominal adipose tissue. Our findings also suggest that 1.2 kb of regulatory sequences present in the hAGT transgene are sufficient to transcriptionally respond to a high-fat diet in an adipose-specific and depot-specific manner.
Stimulation of cardiac sympathetic nerve activity by central angiotensinergic mechanisms in conscious sheep
Authors: Watson Anna M. D. , Mogulkoc Rasim , McAllen Robin M. , May Clive N.
Published: May 2004

Central actions of angiotensin play an important role in cardiovascular control and have been implicated in the pathogenesis of hypertension and heart failure. One feature of centrally or peripherally administered angiotensin is that the bradycardia in response to an acute pressor effect is blunted. It is unknown whether after central angiotensin this is due partly to increased cardiac sympathetic nerve activity (CSNA). We recorded CSNA and arterial pressure in conscious sheep, at least 3 days after electrode implantation. The effects of intracerebroventricular infusions of ANG II (3 nmol/h for 30 min) and artificial cerebrospinal fluid (CSF) (1 ml/h) were determined. The response to intracerebroventricular hypertonic saline (0.6 M NaCl in CSF at 1 ml/h) was examined as there is evidence that hypertonic saline acts via angiotensinergic pathways. Intracerebroventricular angiotensin increased CSNA by 23 ± 7% ( P < 0.001) and mean arterial pressure (MAP) by 7.6 ± 1.2 mmHg ( P < 0.001) but did not significantly change heart rate ( n = 5). During intracerebroventricular ANG II the reflex relation between CSNA and diastolic blood pressure was significantly shifted to the right ( P < 0.01). Intracerebroventricular hypertonic saline increased CSNA (+9.4 ± 6.6%, P < 0.05) and MAP but did not alter heart rate. The responses to angiotensin and hypertonic saline were prevented by intracerebroventricular losartan (1 mg/h). In conclusion, in conscious sheep angiotensin acts within the brain to increase CSNA, despite increased MAP. The increase in CSNA may account partly for the lack of bradycardia in response to the increased arterial pressure. The responses to angiotensin and hypertonic saline were losartan sensitive, indicating they were mediated by angiotensin AT-1 receptors.
Phase-Plane Analysis of Gaze Stabilization to High Acceleration Head Thrusts: A Continuum Across Normal Subjects and Patients With Loss of Vestibular Function
Authors: Peng Grace C. Y. , Zee David S. , Minor Lloyd B.
Published: May 2004

We investigated the vestibulo-ocular reflex (VOR) during high-acceleration, yaw-axis, head rotations in 12 normals and 15 patients with vestibular loss [7 unilateral vestibular deficient (UVD) and 8 bilateral vestibular deficient (BVD)]. We analyzed gaze stabilization within a 200-ms window after head rotation began, using phase planes, which allowed simultaneous analysis of gaze velocity and gaze position. These “gaze planes” revealed critical dynamic information not easily gleaned from traditional gain measurements. We found linear relationships between peak gaze-velocity and peak gaze-position error when normalized to peak head speed and position, respectively. Values fell on a continuum, increasing from normals, to normals tested with very high acceleration (VHA = 10,000–20,000°/s2), to UVD patients during rotations toward the intact side, to UVD patients during rotations toward the lesioned side, to BVD patients. We classified compensatory gaze corrections as gaze-position corrections (GPCs) or gaze-velocity error corrections (GVCs). We defined patients as better-compensated when the value of their end gaze position was low relative to peak gaze position. In the gaze plane this criterion corresponded to relatively stereotyped patterns over many rotations, and appearance of high velocity (100–400°/s) GPCs in the gaze plane ending quadrant (150–200 ms after head movement onset). In less-compensated patients, and normals at VHA, more GVCs were generated, and GPCs were generated only after gaze-velocity error was minimized. These findings suggest that challenges to compensatory vestibular function can be from vestibular deficiency or novel stimuli not previously experienced. Similar patterns of challenge and compensation were observed in both patients with vestibular loss and normal subjects.
Path Integration Deficits during Linear Locomotion after Human Medial Temporal Lobectomy
Authors: Philbeck John W. , Behrmann Marlene , Levy Lucien , Potolicchio Samuel J. , Caputy Anthony J.
Published: May 2004

Animal navigation studies have implicated structures in and around the hippocampal formation as crucial in performing path integration (a method of determining one's position by monitoring internally generated self-motion signals). Less is known about the role of these structures for human path integration. We tested path integration in patients who had undergone left or right medial temporal lobectomy as therapy for epilepsy. This procedure removed approximately 50% of the anterior portion of the hippocampus, as well as the amygdala and lateral temporal lobe. Participants attempted to walk without vision to a previously viewed target 2–6 m distant. Patients with right, but not left, hemisphere lesions exhibited both a decrease in the consistency of path integration and a systematic underregistration of linear displacement (and/or velocity) during walking. Moreover, the deficits were observable even when there were virtually no angular acceleration vestibular signals. The results suggest that structures in the medial temporal lobe participate in human path integration when individuals walk along linear paths and that this is so to a greater extent in right hemisphere structures than left. This information is relevant for future research investigating the neural substrates of navigation, not only in humans (e.g., functional neuroimaging and neuropsychological studies), but also in rodents and other animals.
Direct Current Stimulation over V5 Enhances Visuomotor Coordination by Improving Motion Perception in Humans
Authors: Antal Andrea , Nitsche Michael A. , Kruse Wolfgang , Kincses Tamás Z. , Hoffmann Klaus-Peter , Paulus Walter
Published: May 2004

The primary aim of this study was to determine the extent to which human MT+/V5, an extrastriate visual area known to mediate motion processing, is involved in visuomotor coordination. To pursue this we increased or decreased the excitability of MT+/V5, primary motor, and primary visual cortex by the application of 7 min of anodal and cathodal transcranial direct current stimulation (tDCS) in healthy human subjects while they were performing a visuomotor tracking task involving hand movements. The percentage of correct tracking movements increased specifically during and immediately after cathodal stimulation, which decreases cortical excitability, only when V5 was stimulated. None of the other stimulation conditions affected visuomotor performance. We propose that the improvement in performance caused by cathodal tDCS of V5 is due to a focusing effect on to the complex motion perception conditions involved in this task. This hypothesis was proven by additional experiments: Testing simple and complex motion perception in dot kinetograms, we found that a diminution in excitability induced by cathodal stimulation improved the subject's perception of the direction of the coherent motion only if this was presented among random dots (complex motion perception), and worsened it if only one motion direction was presented (simple movement perception). Our data suggest that area V5 is critically involved in complex motion perception and identification processes important for visuomotor coordination. The results also raise the possibility of the usefulness of tDCS in rehabilitation strategies for neurological patients with visuomotor disorders.
ERPs Reflect Lexical Identification in Word Fragment Priming
Authors: Friedrich Claudia K. , Kotz Sonja A. , Friederici Angela D. , Gunter Thomas C.
Published: May 2004

Behavioral evidence suggests that spoken word recognition involves the temporary activation of multiple entries in a listener's mental lexicon. This phenomenon can be demonstrated in cross-modal word fragment priming (CMWP). In CMWP, an auditory word fragment (prime) is immediately followed by a visual word or pseudoword (target). Experiment 1 investigated ERPs for targets presented in this paradigm. Half of the targets were congruent with the prime (e.g., in the prime-target pair: AM-AMBOSS [anvil]), half were not (e.g., AM-PENSUM [pensum]). Lexical entries of the congruent targets should receive activation from the prime. Thus, lexical identification of these targets should be facilitated. An ERP effect named P350, two frontal negative ERP deflections, and the N400 were sensitive to prime-target congruency. In Experiment 2, the relation of the formerly observed ERP effects to processes in a modality-independent mental lexicon was investigated by presenting primes visually. Only the P350 effect could be replicated across different fragment lengths. Therefore, the P350 is discussed as a correlate of lexical identification in a modality-independent mental lexicon.
Neurobiological Evidence for Abstract Phonological Representations in the Mental Lexicon during Speech Recognition
Authors: Eulitz Carsten , Lahiri Aditi
Published: May 2004

A central issue in speech recognition is how contrastive phonemic information is stored in the mental lexicon. The conventional view assumes that this information is closely related to acoustic properties of speech. Considering that no word is ever pronounced alike twice and that the brain has limited capacities to manage information, an opposing view proposes abstract underspecified representations where not all phonemic features are stored. We examined this proposal using event-related brain potentials, in particular mismatch negativity (MMN), an automatic change detection response in the brain that is sensitive to language-specific phoneme representations. In the current study, vowel pairs were presented to subjects, reversed as standard and deviant. Models not assuming underspecification predict equal MMNs for vowel pairs regardless of the reversal. In contrast, enhanced and earlier MMNs were observed for those conditions where the standard is not phonologically underspecified in the mental representation. This provides the first neurobiological evidence for a featurally underspecified mental lexicon.
Probing the Prerequisites for Motion Blindness
Authors: Niedeggen Michael , Hesselmann Guido , Sahraie Arash , Milders Maarten , Blakemore Colin
Published: May 2004

Neurobiological studies of visual awareness usually focus on the neural events elicited by perceived or nonperceived stimuli but neglect the preexisting conditions that allow (or prevent) conscious perception. We have examined the conditions that lead to temporary motion blindness in a rapid serial visual presentation paradigm, in which subjects have to detect coherent motion in the peripheral stream after a cue (a red fixation point) in the central stream. The failure of awareness depends critically on the occurrence of similar coherent motion events (probes) before the cue. Event-related brain potentials (ERPs) were recorded to track the processing of motion distractors, which determine the prerequisites for this transient deficit. Analysis of motion-evoked responses revealed that there is no progressive reduction in sensitivity in early visual processing. There is, however, a progressive increase in amplitude of a negative wave over the frontal cortex at approximately 250 msec after motion onset and a corresponding reduction of a centro-parietal positivity at approximately 350 msec with an increasing number of distractors. We propose that these nonsensory ERP components reflect a postperceptual frontal gating mechanism that controls the access of visual stimuli to higher order evaluation and conscious detection.
Event-related Brain Potentials Elicited by Morphological, Homographic, Orthographic, and Semantic Priming
Authors: Domínguez Alberto , de Vega Manuel , Barber Horacio
Published: May 2004

The morphological structure of words, in terms of their stem morphemes and affixes, could influence word access and representation in lexical memory. Three experiments were carried out to explore the attributes of event-related potentials evoked by different types of priming. Morphological priming, with pairs of words related by their stem (hijo/hija [son/ daughter]), produced a sustained attenuation (and even a tendency to positivity) of the N400 shown by unrelated words across the three experiments. Homographic priming (Experiment 1), using pairs of words with a superficially similar stem, but without morphological or semantic relation (foco/foca [floodlight/seal]), produced an initial attenuation similar to the morphological pairs, but which rapidly tended to form a delayed N400, due to the impossibility of integration. However, orthographic priming (rasa/rana [flat/frog]) in Experiment 2 does not produce attenuation of the N400 but an effect similar to that of unrelated pairs. Experiment 3 shows that synonyms advance more slowly than morphological pairs to meaning coherence, but finally produce a more positive peak around 600 msec.
Decomposing Components of Task Preparation with Functional Magnetic Resonance Imaging
Authors: Brass Marcel , Cramon D. Yves von
Published: May 2004

It is widely acknowledged that the prefrontal cortex plays a major role in cognitive control processes. One important experimental paradigm for investigating such higher order cognitive control is the task-switching paradigm. This paradigm investigates the ability to switch flexibly between different task situations. In this context, it has been found that participants are able to anticipatorily prepare an upcoming task. This ability has been assumed to reflect endogenous cognitive control. However, it is difficult to isolate task preparation process from task execution using functional magnetic resonance imaging (fMRI). In the present study, we introduce a new experimental manipulation to investigate task preparation with fMRI. By manipulating the number of times a task was prepared, we could demonstrate that the left inferior frontal junction (IFJ) area (near the junction of inferior frontal sulcus and inferior precentral sulcus), the right inferior frontal gyrus, and the right intraparietal sulcus are involved in task preparation. By manipulating the cue-task mapping, we could further show that this activation is not related to cue encoding but to the updating of the relevant task representation. Based on these and previous results, we assume that the IFJ area constitutes a functionally separable division of the lateral prefrontal cortex. Finally, our data suggest that task preparation does not differ for switch and repetition trials in paradigms with a high proportion of switch trials, casting doubt on the assumption that an independent task set reconfiguration process takes place in the preparation interval.