bluuebunny/crossref_metadata_2025 huggingface dataset:
Immediate Response of Mammalian Target of Rapamycin (mTOR)‐Mediated Signalling Following Acute Resistance Exercise in Rat Skeletal Muscle
Authors: Bolster Douglas R. , Kubica Neil , Crozier Stephen J. , Williamson David L. , Farrell Peter A. , Kimball Scot R. , Jefferson Leonard S.
Published: August 2003

The purpose of the present investigation was to determine whether mammalian target of rapamycin (mTOR)‐mediated signalling and some key regulatory proteins of translation initiation are altered in skeletal muscle during the immediate phase of recovery following acute resistance exercise. Rats were operantly conditioned to reach an illuminated bar located high on a Plexiglass cage, such that the animals completed concentric and eccentric contractions involving the hindlimb musculature. Gastrocnemius muscle was extracted immediately after acute exercise and 5, 10, 15, 30 and 60 min of recovery. Phosphorylation of protein kinase B (PKB) on Ser‐473 peaked at 10 min of recovery (282 % of control, P < 0.05) with no significant changes noted for mTOR phosphorylation on Ser‐2448. Eukaryotic initiation factor (eIF) 4E‐binding protein‐1 (4E‐BP1) and S6 kinase‐1 (S6K1), both downstream effectors of mTOR, were altered during recovery as well. 4E‐BP1 phosphorylation was significantly elevated at 10 min (292 %, P < 0.01) of recovery. S6K1 phosphorylation on Thr‐389 demonstrated a trend for peak activation at 10 min following exercise (336 %, P= 0.06) with ribosomal protein S6 phosphorylation being maximally activated at 15 min of recovery (647 %, P < 0.05). Components of the eIF4F complex were enhanced during recovery as eIF4E association with eIF4G peaked at 10 min (292 %, P < 0.05). Events regulating the binding of initiator methionyl‐tRNA to the 40S ribosomal subunit were assessed through eIF2B activity and eIF2α phosphorylation on Ser‐51. No differences were noted with either eIF2B or eIF2α. Collectively, these results provide strong evidence that mTOR‐mediating signalling is transiently upregulated during the immediate period following resistance exercise and this response may constitute the most proximal growth response of the cell.
Arterial Baroreflex Control of Muscle Blood Flow at the Onset of Voluntary Locomotion in Mice
Authors: Masuki Shizue , Nose Hiroshi
Published: August 2003

To assess the role of arterial baroreflex control in muscle blood flow (MBF) and voluntary locomotion, mean arterial pressure (MAP), MBF, and electromyograms (EMGs) were measured in freely moving mice before (CNT) and after blocking the afferent or efferent pathway of arterial baroreflexes, carotid sinus denervation (CSD), or intraperitoneal administration of phentolamine (BLK), respectively. MAP was measured through a catheter placed in the femoral artery. MBF was measured with a needle‐type laser‐Doppler flowmeter and recorded through a low‐pass filter with an edge frequency of 0.1 Hz. The frequency and duration of locomotion were judged from EMG recordings in the hindlimb. These probes were implanted at least 2 days before the measurements. Muscle vascular conductance (MVC = MBF/MAP) in all groups started to rise within 1 s after the onset of locomotion, but the increasing rate in CSD and BLK was significantly higher than in CNT for the first 9 s (P < 0.001). MAP in CSD and BLK significantly decreased below the baseline within 1 s and this was highly correlated with the increase in MVC for the first 9 s (R2= 0.842, P < 0.001), whereas MAP in CNT increased significantly 8 s after the onset of locomotion. Although the total period of movement in a free‐moving state for 60 min was not significantly different between CNT and CSD (P > 0.1), the frequency of movement with a short duration of 0.1‐0.4 min was higher in CSD than in CNT (P < 0.001), which was highly correlated with the reduction in MAP accompanying each period of movement (R2= 0.883, P < 0.01). These results suggest that arterial baroreflexes suppress vasodilatation in contracting muscle to maintain MAP at the onset of voluntary locomotion, and are necessary to continue a given duration of locomotion in mice.
P2X2 subunits contribute to fast synaptic excitation in myenteric neurons of the mouse small intestine
Authors: Ren Jianhua , Bian Xiaochun , DeVries Matthew , Schnegelsberg Birthe , Cockayne Debra A. , Ford Anthony P. D. W. , Galligan James J.
Published: August 2003

P2X receptors are ATP‐gated cation channels composed of one or more of seven different subunits. ATP acts at P2X receptors to contribute to fast excitatory postsynaptic potentials (fEPSPs) in myenteric neurons but the subunit composition of enteric P2X receptors is unknown. These studies used tissues from P2X2 wild‐type (P2X2+/+) and P2X2 gene knockout (P2X2‐/‐) mice to investigate the role of this subunit in enteric neurotransmission. Intracellular electrophysiological methods were used to record synaptic and drug‐induced responses from ileal myenteric neurons in vitro. Drug‐induced longitudinal muscle contractions and peristaltic contractions of ileal segments were also studied in vitro. Gastrointestinal transit was measured as the progression in 30 min of a liquid radioactive marker administered by gavage to fasted mice. RT‐PCR analysis of mRNA from intestinal tissues and data from immunohistochemical studies verified P2X2 gene deletion. The fEPSPs recorded from S neurons in tissues from P2X2+/+ mice were reduced by mecamylamine (nicotinic cholinergic receptor antagonist) and PPADS (P2X receptor antagonist). The fEPSPs recorded from S neurons from P2X2−/− mice were unaffected by PPADS but were blocked by mecamylamine. ATP depolarized S and AH neurons from P2X2+/+ mice. ATP depolarized AH but not S neurons from P2X2‐/‐ mice. α,β‐Methylene ATP (α,β‐mATP)(an agonist at P2X3 subunit‐containing receptors) did not depolarize S neurons but it did depolarize AH neurons in P2X2+/+ and P2X2‐/‐ mice. Peristalsis was inhibited in ileal segments from P2X2‐/‐ mice but longitudinal muscle contractions caused by nicotine and bethanechol were similar in segments from P2X2+/+ and P2X2‐/‐ mice. Gastrointestinal transit was similar in P2X2+/+ and P2X2‐/‐ mice. It is concluded that P2X2 homomeric receptors contribute to fEPSPs in neural pathways underlying peristalsis studied in vitro.
Insulin‐like growth factor‐1 prevents age‐related decrease in specific force and intracellular Ca2+ in single intact muscle fibres from transgenic mice
Authors: González Estela , Messi María Laura , Zheng Zhenlin , Delbono Osvaldo
Published: August 2003

In the present work we test the hypothesis that sustained transgenic overexpression of insulin‐like growth factor‐1 (IGF‐1) in skeletal muscle prevents age‐related decreases in myoplasmic Ca2+ concentration and consequently in specific force in single intact fibres from the flexor digitorum brevis (FDB) muscle from the mouse. Measurements of IGF‐1 concentration in FDB muscle showed higher levels in transgenic than in wild‐type mice at all ages. The specific tetanic force decreased significantly in single muscle fibres from old (286 ± 22 kPa) compared to young wild‐type (455 ± 28 kPa), young transgenic (423 ± 43 kPa), and old transgenic mice (386 ± 15 kPa) (P < 0.05). These results are consistent with measurements in whole FDB muscles. The peak Ca2+ concentration values in response to prolonged stimulation were: 1.47 ± 0.15, 1.70 ± 0.29, 0.97 ± 0.13 and 1.7 ± 0.22 μm, in fibres from young wild‐type, young transgenic, old wild‐type and old transgenic mice, respectively. The effects of caffeine on FDB fibres support the conclusion that the age‐related decline in peak myoplasmic Ca2+ and specific force is not explained by sarcoplasmic reticulum Ca2+ depletion. Immunohistochemistry in muscle cross‐sections was performed to determine whether age and/or IGF‐1 overexpression induce changes in fibre type composition. The relative percentages of type IIa, IIx and I myosin heavy chain (MHC) isoforms did not change significantly with age or genotype. Therefore, IGF‐1 prevents age‐related decline in peak intracellular Ca2+ and specific force in a muscle that does not exhibit changes in fibre type composition with senescence.
Simple‐sugar meals target GLUT2 at enterocyte apical membranes to improve sugar absorption: a study in GLUT2‐null mice
Authors: Gouyon F. , Caillaud L. , Carrière V. , Klein C. , Dalet V. , Citadelle D. , Kellett G. L. , Thorens B. , Leturque A. , Brot‐Laroche E.
Published: August 2003

The physiological significance of the presence of GLUT2 at the food‐facing pole of intestinal cells is addressed by a study of fructose absorption in GLUT2‐null and control mice submitted to different sugar diets. Confocal microscopy localization, protein and mRNA abundance, as well as tissue and membrane vesicle uptakes of fructose were assayed. GLUT2 was located in the basolateral membrane of mice fed a meal devoid of sugar or containing complex carbohydrates. In addition, the ingestion of a simple sugar meal promoted the massive recruitment of GLUT2 to the food‐facing membrane. Fructose uptake in brush‐border membrane vesicles from GLUT2‐null mice was half that of wild‐type mice and was similar to the cytochalasin B‐insensitive component, i.e. GLUT5‐mediated uptake. A 5 day consumption of sugar‐rich diets increased fructose uptake fivefold in wild‐type tissue rings when it only doubled in GLUT2‐null tissue. GLUT5 was estimated to contribute to 100 % of total uptake in wild‐type mice fed low‐sugar diets, falling to 60 and 40 % with glucose and fructose diets respectively; the complement was ensured by GLUT2 activity. The results indicate that basal sugar uptake is mediated by the resident food‐facing SGLT1 and GLUT5 transporters, whose mRNA abundances double in long‐term dietary adaptation. We also observe that a large improvement of intestinal absorption is promoted by the transient recruitment of food‐facing GLUT2, induced by the ingestion of a simple‐sugar meal. Thus, GLUT2 and GLUT5 could exert complementary roles in adapting the absorption capacity of the intestine to occasional or repeated loads of dietary sugars.
Contribution of Ca2+‐dependent conductances to membrane potential fluctuations of medullary respiratory neurons of newborn rats in vitro
Authors: Onimaru Hiroshi , Ballanyi Klaus , Homma Ikuo
Published: August 2003

Ca2+‐dependent conductances were studied in respiratory interneurons in the brainstem‐spinal cord preparation of newborn rats. ω‐Conotoxin‐GVIA attenuated evoked postsynaptic potentials, spontaneous or evoked inspiratory spinal nerve activity and blocked spike afterhyperpolarization. Furthermore, ω‐conotoxin‐GVIA augmented rhythmic drive potentials of pre‐inspiratory and inspiratory neurons and increased respiratory‐related spike frequency of pre‐inspiratory cells with no effect on inspiratory hyperpolarization. In contrast, ω‐agatoxin‐IVA depressed drive potentials of pre‐inspiratory and inspiratory neurons and attenuated inspiratory hyperpolarization and spike frequency of pre‐inspiratory cells. It did not affect spike shape and exerted only minor, non‐significant, attenuating effects on spontaneous or evoked nerve bursts or evoked postsynaptic potentials. Nifedipine diminished drive potentials and spike frequency of pre‐inspiratory neurons and shortened drive potentials in some cells. ω‐Conotoxin‐MVIIC attenuated drive potentials and intraburst firing rate of pre‐inspiratory neurons and decreased substantially respiratory frequency. Respiratory rhythm disappeared following combined application of ω‐conotoxin‐GVIA, ω‐conotoxin‐MVIIC, ω‐agatoxin‐IVA and nifedipine. Apamin potentiated drive potentials and abolished spike afterhyperpolarization, whereas charybdotoxin and tetraethylammonium prolonged spike duration without effect on shape of drive potentials. The results show that specific sets of voltage‐activated L‐, N‐ and P/Q‐type Ca2+ channels determine the activity of particular subclasses of neonatal respiratory neurons, whereas SK‐ and BK‐type K+ channels attenuate drive potentials and shorten spikes, respectively, independent of cell type. We hypothesize that modulation of spontaneous activity of pre‐inspiratory neurons via N‐, L‐ and P/Q‐type Ca2+ channels is important for respiratory rhythm or pattern generation.
In vivo determination of steric and electrostatic exclusion of albumin in rat skin and skeletal muscle
Authors: Gyenge Christina C. , Tenstad Olav , Wiig Helge
Published: August 2003

In order to estimate the magnitude of electrostatic exclusion provided by the fixed negative charges of the skin and muscle interstitia of rat in vivo we measured the distribution volumes of two differently charged albumin probes within these tissues. An implanted osmotic pump was used to reach and maintain a steady‐state extracellular concentration of a mixture containing two iodine‐labelled probes: a charged‐modified human serum albumin, cHSA (i.e. a positive probe, isoelectirc point (pI)= 7.6) and a native human serum albumin, HSA (i.e. a normally charged, negative probe, pI = 5.0). Steady‐state tissue concentrations were achieved after intravenous infusion of probes for 5–7 days. At the end of this period the animals were nephrectomized and a bolus of 51Cr‐EDTA was administered for estimating the extracellular volume. Plasma volumes were measured as 5‐min distribution volume of 125I‐HSA in separate experiments. The steady‐state interstitial fluid concentrations of all probes were determined using nylon wicks implanted postmortem. Calculations of labelled probes were made for interstitial fluid volumes (Vi), extravascular albumin distribution volumes (Vav,a) and relative interstitial excluded volume fractions (Vex,a/Vi). We found that the positive probe is excluded from a significantly smaller fraction of the interstitium. Specifically, the average relative albumin exclusion fractions obtained were: 16% and 26% in skeletal muscle and 30% and 40% in skin, for cHSA and HSA, respectively. On average, the fixed negative charges of the interstitium are responsible for about 40% of the total albumin exclusion in skeletal muscle and 25% in the whole skin tissue and thus, contribute significantly to volume exclusion in these tissues.
Requirement of β‐adrenergic receptor activation and protein synthesis for LTP‐reinforcement by novelty in rat dentate gyrus
Authors: Straube Thomas , Korz Volker , Balschun Detlef , Uta Frey Julietta
Published: August 2003

Long‐term potentiation (LTP) is supposed to be a cellular mechanism involved in memory formation. Similar to distinct types of memory formation, LTP can be separated into a protein synthesis‐independent early phase (early‐LTP) and a protein synthesis‐dependent late phase (late‐LTP). An important question is whether the transformation from early‐ into late‐LTP can be elicited by behavioural conditions such as the attention to novel events. Therefore, we investigated the effect of exploration of a novel environment (novelty‐exploration) on subsequently induced early‐LTP in the dentate gyrus of freely moving rats. While a delay of 60 min between exploration onset and LTP induction had no effect, intervals of 30 or 15 min led to a reinforcement of early‐ to late‐LTP. Exploration of a familiar environment failed to prolong LTP maintenance. The novelty‐induced LTP reinforcement was blocked when the translation inhibitor anisomycin or the β‐adrenergic antagonist propranolol were applied intracerebroventricularly before exploration onset. These findings support the hypothesis that the synergistic interplay of novelty‐triggered noradrenergic activity and weak tetanic stimulation promotes the synthesis of certain proteins that are required for late‐LTP. Such a cellular mechanism may underlie novelty‐dependent enhancement of memory formation.
The effects of asphyxia on renal function in fetal sheep at midgestation
Authors: O'Connell A. E. , Boyce A. C. , Lumbers E. R. , Gibson K. J.
Published: August 2003

To determine whether damage to the fetal kidneys plays a role in the formation of hydrops fetalis following a severe asphyxial episode, six chronically catheterised fetal sheep, at 0.6 gestation (90 days; term 150 days), were subjected to 30 min of complete umbilical cord occlusion. During the occlusion period, mean arterial pressure, heart rate and renal blood flow decreased (P < 0.001). There were falls in arterial pH and PO2 and a rise in PCO2 (P < 0.001). Urine flow rate decreased (P < 0.005), as did the excretion rates of sodium and osmoles (P < 0.05). However, by 60 min after release of occlusion, urine flow rate was similar to control values. By the end of day 1, most renal variables returned to normal. At post‐mortem, 72 h after occlusion, all asphyxiated fetuses showed gross signs of hydrops. Body weight was higher (P < 0.05) due to fluid accumulation in the peritoneal (P < 0.001) and pleural cavities (P < 0.05) as well as subcutaneously (P < 0.05). Amniotic/allantoic fluid volume was increased (P < 0.05). Kidney histology was normal except for clusters of apoptotic cells in some proximal tubules. In conclusion, this severe asphyxial episode caused surprisingly little damage to the kidney and the changes in renal function were very transient. Thus renal damage was not important in the development of hydrops. Possibly, the midgestation fetal kidney has a limited capacity to increase urinary salt and water excretion in response to increased fluid delivery across the placenta.
Hyperpolarisation Rectification in Cat Lateral Geniculate Neurons Modulated by Intact Corticothalamic Projections
Authors: Nita D. A. , Steriade M. , Amzica F.
Published: August 2003

The intrinsic properties of thalamic neurons are influenced by synaptic activities in ascending pathways and corticofugal projections, as well as by the actions of neurotransmitters released by generalised modulatory systems. We focused on the effects of corticothalamic projections on the hyperpolarisation‐activated cation current Ih. Intracellular recordings of thalamocortical neurons in the dorsal lateral geniculate (dLG) nucleus were performed in cats under ketamine‐xylazine anaesthesia. At variance with the conventional way of recording intracellularly from thalamic neurons after partial or total ablation of the grey and white matter overlying the dLG, we preserved intact corticothalamic neuronal loops. Stimulating electrodes inserted into the optic tract and light‐emitting‐diodes as photic stimulation were used to identify the dLG neurons. The expression of the depolarising sag due to Ih depended on the state of cortical networks. Thalamic dLG Ih, induced by hyperpolarising current steps, was detected during the periods of cortical disfacilitation that occur during the cortical slow (< 1 Hz) oscillation, whereas Ih was absent during the active (depolarised) periods. The possibility that the excitatory corticothalamic projections could preclude the generation of the Ih was tested by applying a concentrated K+ solution (3 M) to the primary visual cortex. The same dLG neurons that did not display Ih before application of K+ were able to produce hyperpolarisation‐activated depolarising sags during K+‐induced cortical depression. Our data suggest that the thalamic clock‐like delta oscillation, which results from an interplay between Ih and the low‐threshold calcium current (IT), as described in preparations without cerebral cortex, is prevented in dLG neurons when corticothalamic loops are intact.
Modulation of Whole‐Cell Currents in Plasmodium Falciparum‐Infected Human Red Blood Cells by Holding Potential and Serum
Authors: Staines Henry M. , Powell Trevor , Clive Ellory J. , Egée Stéphane , Lapaix Franck , Decherf Gaëtan , Thomas Serge L. Y. , Duranton Christophe , Lang Florian , Huber Stephan M.
Published: August 2003

Recent electrophysiological studies have identified novel ion channel activity in the host plasma membrane of Plasmodium falciparum‐infected human red blood cells (RBCs). However, conflicting data have been published with regard to the characteristics of induced channel activity measured in the whole‐cell configuration of the patch‐clamp technique. In an effort to establish the reasons for these discrepancies, we demonstrate here two factors that have been found to modulate whole‐cell recordings in malaria‐infected RBCs. Firstly, negative holding potentials reduced inward currents (i.e. at negative potentials), although this result was highly complex. Secondly, the addition of human serum increased outward currents (i.e. at positive potentials) by approximately 4‐fold and inward currents by approximately 2‐fold. These two effects may help to resolve the conflicting data in the literature, although further investigation is required to understand the underlying mechanisms and their physiological relevance in detail.
Contractile effects of the exchange of cardiac troponin for fast skeletal troponin in rabbit psoas single myofibrils
Authors: Piroddi N. , Tesi C. , Pellegrino M. A. , Tobacman L. S. , Homsher E. , Poggesi C.
Published: August 2003

The effects of the removal of fast skeletal troponin C (fsTnC) and its replacement by cardiac troponin C (cTnC) and the exchange of fast skeletal troponin (fsTn) for cardiac troponin (cTn) were measured in rabbit fast skeletal myofibrils. Electrophoretic analysis of myofibril suspensions indicated that replacement of fsTnC or exchange of fsTn with cTnC or cTn was about 90% complete in the protocols used. Mechanical measurements in single myofibrils, which were maximally activated by fast solution switching, showed that replacement of fsTnC with cTnC reduced the isometric tension, the rate of tension rise following a step increase in Ca2+ (kact), and the rate of tension redevelopment following a quick release and restretch (ktr), but had no effect on the kinetics of the fall in tension when the concentration of inorganic phosphate (Pi) was abruptly increased (kPi(+)). These data suggest that the chimeric protein produced by cTnC replacement in fsTn alters those steps controlling the weak‐to‐strong crossbridge attachment transition. Inefficient signalling within the chimeric troponin may cause these changes. However, replacement of fsTn by cTn had no effect on maximal isometric tension, kact or ktr, suggesting that these mechanics are largely determined by the isoform of the myosin molecule. Replacement of fsTn by cTn, on the other hand, shifted the pCa50 of the pCa‐tension relationship from 5.70 to 6.44 and reduced the Hill coefficient from 3.3 to 1.4, suggesting that regulatory protein isoforms primarily alter Ca2+ sensitivity and the cooperativity of the force‐generating mechanism.
Role of inhibitory amino acids in control of hypoglossal motor outflow to genioglossus muscle in naturally sleeping rats
Authors: Morrison Janna L. , Sood Sandeep , Liu Hattie , Park Eileen , Liu Xia , Nolan Philip , Horner Richard L.
Published: August 2003

The hypoglossal motor nucleus innervates the genioglossus (GG) muscle of the tongue, a muscle that helps maintain an open airway for effective breathing. Rapid‐eye‐movement (REM) sleep, however, recruits powerful neural mechanisms that can abolish GG activity even during strong reflex stimulation such as by hypercapnia, effects that can predispose to sleep‐related breathing problems in humans. We have developed an animal model to chronically manipulate neurotransmission at the hypoglossal motor nucleus using in vivo microdialysis in freely behaving rats. This study tests the hypothesis that glycine receptor antagonism at the hypoglossal motor nucleus, either alone or in combination with GABAA receptor antagonism, will prevent suppression of GG activity in natural REM sleep during room air and CO2‐stimulated breathing. Rats were implanted with electroencephalogram and neck muscle electrodes to record sleep–wake states, and GG and diaphragm electrodes for respiratory muscle recording. Microdialysis probes were implanted into the hypoglossal motor nucleus for perfusion of artificial cerebrospinal fluid (ACSF) and strychnine (glycine receptor antagonist, 0.1 mm) either alone or combined with bicuculline (GABAA antagonist, 0.1 mm) during room air and CO2‐stimulated breathing. Compared to ACSF controls, glycine receptor antagonism at the hypoglossal motor nucleus increased respiratory‐related GG activity in room air (P= 0.010) but not hypercapnia (P= 0.221). This stimulating effect of strychnine in room air did not depend on the prevailing sleep–wake state (P= 0.625) indicating removal of a non‐specific background inhibitory glycinergic tone. Nevertheless, GG activity remained minimal in those REM sleep periods without phasic twitches in GG muscle, with GG suppression from non‐REM (NREM) sleep being > 85% whether ACSF or strychnine was at the hypoglossal motor nucleus or the inspired gas was room air or 7% CO2. While GG activity was minimal in these REM sleep periods, there was a small but measurable increase in GG activity after strychnine (P < 0.05). GG activity was also minimal, and effectively abolished, in the REM sleep periods without GG twitches with combined glycine and GABAA receptor antagonism at the hypoglossal motor nucleus. We conclude that these data in freely behaving rats confirm that inhibitory glycine and GABAA receptor mechanisms are present at the hypoglossal motor nucleus and are tonically active, but that such inhibitory mechanisms make only a small contribution to the marked suppression of GG activity and reflex responses observed in periods of natural REM sleep.
Single-Molecule Kinetics of λ Exonuclease Reveal Base Dependence and Dynamic Disorder
Authors: van Oijen Antoine M. , Blainey Paul C. , Crampton Donald J. , Richardson Charles C. , Ellenberger Tom , Xie X. Sunney
Published: August 2003

We used a multiplexed approach based on flow-stretched DNA to monitor the enzymatic digestion of λ-phage DNA by individual bacteriophage λ exonuclease molecules. Statistical analyses of multiple single-molecule trajectories observed simultaneously reveal that the catalytic rate is dependent on the local base content of the substrate DNA. By relating single-molecule kinetics to the free energies of hydrogen bonding and base stacking, we establish that the melting of a base from the DNA is the rate-limiting step in the catalytic cycle. The catalytic rate also exhibits large fluctuations independent of the sequence, which we attribute to conformational changes of the enzyme-DNA complex.
Polarization-Modulated Smectic Liquid Crystal Phases
Authors: Coleman D. A. , Fernsler J. , Chattham N. , Nakata M. , Takanishi Y. , Körblova E. , Link D. R. , Shao R.-F. , Jang W. G. , Maclennan J. E. , Mondainn-Monval O. , Boyer C. , Weissflog W. , Pelzl G. , Chien L.-C. , Zasadzinski J. , Watanabe J. , Walba D. M. , Takezoe H. , Clark N. A.
Published: August 2003

Any polar-ordered material with a spatially uniform polarization field is internally frustrated: The symmetry-required local preference for polarization is to be nonuniform, i.e., to be locally bouquet-like or “splayed.” However, it is impossible to achieve splay of a preferred sign everywhere in space unless appropriate defects are introduced into the field. Typically, in materials like ferroelectric crystals or liquid crystals, such defects are not thermally stable, so that the local preference is globally frustrated and the polarization field remains uniform. Here, we report a class of fluid polar smectic liquid crystals in which local splay prevails in the form of periodic supermolecular-scale polarization modulation stripes coupled to layer undulation waves. The polar domains are locally chiral, and organized into patterns of alternating handedness and polarity. The fluid-layer undulations enable an extraordinary menagerie of filament and planar structures that identify such phases.
Control Mechanism of the Circadian Clock for Timing of Cell Division in Vivo
Authors: Matsuo Takuya , Yamaguchi Shun , Mitsui Shigeru , Emi Aki , Shimoda Fukuko , Okamura Hitoshi
Published: August 2003

Cell division in many mammalian tissues is associated with specific times of day, but just how the circadian clock controls this timing has not been clear. Here, we show in the regenerating liver (of mice) that the circadian clock controls the expression of cell cycle–related genes that in turn modulate the expression of active Cyclin B1-Cdc2 kinase, a key regulator of mitosis. Among these genes, expression of
wee1
was directly regulated by the molecular components of the circadian clockwork. In contrast, the circadian clockwork oscillated independently of the cell cycle in single cells. Thus, the intracellular circadian clockwork can control the cell-division cycle directly and unidirectionally in proliferating cells.
Rapid Manufacturing of Aluminum Components
Authors: Sercombe T. B. , Schaffer G. B.
Published: August 2003

A manufacturing technique for the production of aluminum components is described. A resin-bonded part is formed by a rapid prototyping technique and then debound and infiltrated by a second aluminum alloy under a nitrogen atmosphere. During thermal processing, the aluminum reacts with the nitrogen and is partially transformed into a rigid aluminum nitride skeleton, which provides the structural rigidity during infiltration. The simplicity and rapidity of this process in comparison to conventional production routes, combined with the ability to fabricate complicated parts of almost any geometry and with high dimensional precision, provide an additional means to manufacture aluminum components.
A Gene-Coexpression Network for Global Discovery of Conserved Genetic Modules
Authors: Stuart Joshua M. , Segal Eran , Koller Daphne , Kim Stuart K.
Published: August 2003

To elucidate gene function on a global scale, we identified pairs of genes that are coexpressed over 3182 DNA microarrays from humans, flies, worms, and yeast. We found 22,163 such coexpression relationships, each of which has been conserved across evolution. This conservation implies that the coexpression of these gene pairs confers a selective advantage and therefore that these genes are functionally related. Manyof these relationships provide strong evidence for the involvement of new genes in core biological functions such as the cell cycle, secretion, and protein expression. We experimentallyconfirmed the predictions implied bysome of these links and identified cell proliferation functions for several genes. By assembling these links into a gene-coexpression network, we found several components that were animal-specific as well as interrelationships between newly evolved and ancient modules.
Preparation of a Pure Molecular Quantum Gas
Authors: Herbig Jens , Kraemer Tobias , Mark Michael , Weber Tino , Chin Cheng , Nägerl Hanns-Christoph , Grimm Rudolf
Published: August 2003

An ultracold molecular quantum gas is created by application of a magnetic field sweep across a Feshbach resonance to a Bose-Einstein condensate of cesium atoms. The ability to separate the molecules from the atoms permits direct imaging of the pure molecular sample. Magnetic levitation enables study of the dynamics of the ensemble on extended time scales. We measured ultralow expansion energies in the range of a few nanokelvin for a sample of 3000 molecules. Our observations are consistent with the presence of a macroscopic molecular matter wave.
Functional Analysis of Mosquito-Borne Flavivirus Conserved Sequence Elements within 3′ Untranslated Region of West Nile Virus by Use of a Reporting Replicon That Differentiates between Viral Translation and RNA Replication
Authors: Lo Michael K. , Tilgner Mark , Bernard Kristen A. , Shi Pei-Yong
Published: August 2003

We have developed a reporting replicon of West Nile virus (WNV) that could be used to quantitatively distinguish viral translation and RNA replication. A
Renilla
luciferase (Rluc) gene was fused in-frame with the open reading frame of a subgenomic replicon in the position where the viral structural region was deleted, resulting in RlucRep. Transfection of BHK cells with RlucRep RNA yielded two distinctive Rluc signal peaks, one between 2 and 10 h and the other after 26 h posttransfection. By contrast, only the 2- to 10-h Rluc signal peak was observed in cells transfected with a mutant replicon containing an inactivated viral polymerase NS5 (RlucRep-NS5mt). Immunofluorescence and real-time reverse transcriptase PCR assays showed that the levels of viral protein expression and RNA replication increased in cells transfected with the RlucRep but not in those transfected with the RlucRep-NS5mt. These results suggest that the Rluc signal that occurred at 2 to 10 h posttransfection reflects viral translation of the input replicon, while the Rluc activity after 26 h posttransfection represents RNA replication. Using this system, we showed that mutations of conserved sequence (CS) elements within the 3′ untranslated region of the mosquito-borne flaviviruses did not significantly affect WNV translation but severely diminished or completely abolished RNA replication. Mutations of CS1 that blocked the potential base pairing with a conserved sequence in the 5′ region of the capsid gene (5′CS) abolished RNA replication. Restoration of the 5′CS-CS1 interaction rescued viral replication. Replicons containing individual deletions of CS2, repeated CS2 (RCS2), CS3, or RCS3 were viable, but their RNA replication was dramatically compromised. These results demonstrate that genome cyclization through the 5′CS-CS1 interaction is essential for WNV RNA replication, whereas CS2, RCS2, CS3, and RCS3 facilitate, but are dispensable for, WNV replication.
SR-Related Protein TAXREB803/SRL300 Is an Important Cellular Factor for the Transactivational Function of Human T-Cell Lymphotropic Virus Type 1 Tax
Authors: Youn Hwang-Geum , Matsumoto Jun , Tanaka Yuetsu , Shimotohno Kunitada
Published: August 2003

Expression of the human T-cell lymphotropic virus type 1 (HTLV-1) genes is transcriptionally activated by the cognate oncoprotein Tax which enhances the binding of the cyclin AMP-responsive element binding protein (CREB) to the Tax responsive element (TxRE) located in its long terminal repeat (LTR). TxRE is highly homologous to the cyclic AMP-responsive element (CRE) except for the GC-rich sequence flanking the CRE. We cloned the cDNA for a cellular factor, TAXREB803, of which the DNA-binding domain bound to TxRE and the binding was dependent on the 3′ GC-rich sequence in TxRE. TAXREB803 is an SR-related protein composed of 2,752 amino acids including numerous arginine/serine (RS) motifs. TAXREB803 enhanced both the Tax dependent transcription and the CREB binding to TxRE in cooperation with Tax. The interaction of TAXREB803 and Tax was detected by coimmunoprecipitation assays as well as by indirect immunofluorescence assays. Significantly, Tax transactivation for the HTLV-1 LTR decreased dramatically when the expression level of the endogenous TAXREB803 was suppressed by the small interfering RNA. These results suggest that TAXREB803 functions as a transcriptional coactivator for Tax and plays a critical role in the expression of HTLV-1 genes.
Theoretical Design of a Gene Therapy To Prevent AIDS but Not Human Immunodeficiency Virus Type 1 Infection
Authors: Weinberger Leor S. , Schaffer David V. , Arkin Adam P.
Published: August 2003

Recent reports confirm that, due to the presence of long-lived, latently infected cell populations, eradication of human immunodeficiency virus type 1 (HIV-1) from infected patients by using antiretroviral drugs will be exceedingly difficult. An alternative to virus eradication may be to use gene therapy to induce a pseudo-latent state in virus-producing cells, thus transforming HIV-1 into a lifelong, but manageable, virus. Conditionally replicating HIV-1 (crHIV-1) gene therapy vectors provide an avenue for subduing HIV-1 expression in infected cells (by creating a parasite, crHIV-1, of the parasite HIV-1), potentially reducing the HIV-1 set point and delaying AIDS onset. Development of crHIV-1 vectors has proceeded in vitro, but the requirements for a crHIV-1 vector to proliferate and persist in vivo have not been explored. We expand a widely accepted mathematical model of HIV-1 in vivo dynamics to include a crHIV-1 gene therapy virus and derive a simple criterion for designing crHIV-1 viruses that will persist in vivo. The model introduces only two new parameters—HIV-1 inhibition and crHIV-1 production—and both can be experimentally engineered and controlled. Analysis demonstrates that crHIV-1 gene therapy can indefinitely reduce HIV-1 set point to levels comparable to those achieved with highly active antiretroviral therapy, provided crHIV-1 production is more efficient than HIV-1. Paradoxically, highly efficient therapeutic inhibition of HIV-1 was found to be disadvantageous. Thus, the field may benefit by shifting the search for more potent antiviral genes toward engineering optimized therapy viruses that package ultraefficiently while downregulating viral production moderately.
Effect of Anti-CXCL10 Monoclonal Antibody on Herpes Simplex Virus Type 1 Keratitis and Retinal Infection
Authors: Carr Daniel J. J. , Chodosh James , Ash John , Lane Thomas E.
Published: August 2003

The inflammatory response to acute ocular herpes simplex virus type 1 (HSV-1) infection in mice involves the innate and adaptive immune response, with an associated increase in the secretion of chemokines, including CXCL10 (interferon-inducible protein 10 kDa [IP-10]). Neutralizing antibodies to mouse CXCL10 were used to determine the role of CXCL10 during the acute phase of HSV-1 ocular infection. Treatment of HSV-1-infected mice with antibody to CXCL10 significantly reduced CXCL10 levels in the eye and trigeminal ganglion and reduced mononuclear cell infiltration into the corneal stroma. These results coincided with reduced ICAM-1 and CXCR3 transcript expression, macrophage inflammatory protein-1α and CXCL10 levels, and corneal pathology but increased viral titers in the stroma and trigeminal ganglion. Progression of the virus from the corneal stroma to the retina during acute infection was significantly hindered in anti-CXCL10-treated mice. In addition, colocalization of viral antigen with infiltrating leukocytes in the iris and retina during acute infection suggests that one means by which HSV-1 traffics to the retina involves inflammatory cells (primarily CD11b+cells). Collectively, the results suggest that CXCL10 expression in the eye initially orchestrates the inflammatory response to acute HSV-1 infection, which facilitates the spread of the virus to other restricted sites within the eye.
Distinct Cycling CD4+- and CD8+-T-Cell Profiles during the Asymptomatic Phase of Simian Immunodeficiency Virus SIVmac251 Infection in Rhesus Macaques
Authors: Monceaux V. , Fang R. Ho Tsong , Cumont M. C. , Hurtrel B. , Estaquier J.
Published: August 2003

Elevated CD4 T-cell turnover may lead to the exhaustion of the immune system during human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) infections. However, this hypothesis remains controversial. Most studies of this subject have concerned the blood, and information about the lymph nodes is rare and controversial. We used Ki67 expression to measure cycling T cells in the blood and lymph nodes of uninfected macaques and of macaques infected with a pathogenic SIVmac251 strain or with a nonpathogenic SIVmac251Δnef clone. During the asymptomatic phase of infection, the number of cycling CD8+T cells progressively increased (two- to eightfold) both in the blood and in the lymph nodes of macaques infected with SIVmac251. This increase was correlated with viral replication and the progression to AIDS. In contrast, no increases in the numbers of cycling CD4+T cells were found in the blood or lymph nodes of macaques infected with the pathogenic SIVmac251 strain in comparison with SIVmac251Δnef-infected or healthy macaques during this chronic phase. However, the lymph nodes of pre-AIDS stage SIVmac251-infected macaques contained more cycling CD4+T cells (low baseline CD4+-T-cell counts in the blood). Taken together, these results show that the profiles of CD4+- and CD8+-T-cell dynamics are distinct both in the lymph nodes and blood and suggest that higher CD4+-T-cell proliferation at the onset of AIDS may lead to the exhaustion of the immune system.
T Cells Cause Acute Immunopathology and Are Required for Long-Term Survival in Mouse Adenovirus Type 1-Induced Encephalomyelitis
Authors: Moore Martin L. , Brown Corrie C. , Spindler Katherine R.
Published: August 2003

Infection of adult C57BL/6 (B6) mice with mouse adenovirus type 1 (MAV-1) results in dose-dependent encephalomyelitis. Utilizing immunodeficient mice, we analyzed the roles of T cells, T-cell subsets, and T-cell-related functions in MAV-1-induced encephalomyelitis. T cells, major histocompatibility complex (MHC) class I, and perforin contributed to acute disease signs at 8 days postinfection (p.i.). Acute MAV-1-induced encephalomyelitis was absent in mice lacking T cells and in mice lacking perforin. Mice lacking α/β T cells had higher levels of infectious MAV-1 at 8 days, 21 days, and 12 weeks p.i., and these mice succumbed to MAV-1-induced encephalomyelitis at 9 to 16 weeks p.i. Thus, α/β T cells were required for clearance of MAV-1. MAV-1 was cleared in mice lacking perforin, MHC class I or II, CD4+T cells, or CD8+T cells. Our results are consistent with a model in which either CD8+or CD4+T cells are sufficient for clearance of MAV-1. Furthermore, perforin contributed to MAV-1 disease but not viral clearance. We have established two critical roles for T cells in MAV-1-induced encephalomyelitis. T cells caused acute immunopathology and were required for long-term host survival of MAV-1 infection.
Heparan Sulfates and Coxsackievirus-Adenovirus Receptor: Each One Mediates Coxsackievirus B3 PD Infection
Authors: Zautner A. E. , Körner U. , Henke A. , Badorff C. , Schmidtke M.
Published: August 2003

Amino acid exchanges in the virus capsid protein VP1 allow the coxsackievirus B3 variant PD (CVB3 PD) to replicate in decay accelerating factor (DAF)-negative and coxsackievirus-adenovirus receptor (CAR)-negative cells. This suggests that molecules other than DAF and CAR are involved in attachment of this CVB3 variant to cell surfaces. The observation that productive infection associated with cytopathic effect occurred in Chinese hamster ovary (CHO-K1) cells, whereas heparinase-treated CHO-K1 cells, glucosaminoglycan-negative pgsA-745, heparan sulfate (HS)-negative pgsD-677, and pgsE-606 cells with significantly reduced N-sulfate expression resist CVB3 PD infection, indicates a critical role of highly sulfated HS. 2-
O
-sulfate-lacking pgsF-17 cells represented the cell line with minimum HS modifications susceptible for CVB3 PD. Inhibition of virus replication in CHO-K1 cells by polycationic compounds, pentosan polysulfate, lung heparin, and several intestinal but not kidney HS supported the hypothesis that CVB3 PD uses specific modified HS for entry. In addition, recombinant human hepatocyte growth factor blocked CVB3 PD infection. However, CAR also mediates CVB3 PD infection, because this CVB3 variant replicates in HS-lacking but CAR-bearing Raji cells, infection could be prevented by pretreatment of cells with CAR antibody, and HS-negative pgsD-677 cells transfected with CAR became susceptible for CVB3 PD. These results demonstrate that the amino acid substitutions in the viral capsid protein VP1 enable CVB3 PD to use specific modified HS as an entry receptor in addition to CAR.
Enhanced Mucosal Immunoglobulin A Response of Intranasal Adenoviral Vector Human Immunodeficiency Virus Vaccine and Localization in the Central Nervous System
Authors: Lemiale Franck , Kong Wing-pui , Akyürek Levent M. , Ling Xu , Huang Yue , Chakrabarti Bimal K. , Eckhaus Michael , Nabel Gary J.
Published: August 2003

Replication-defective adenovirus (ADV) vectors represent a promising potential platform for the development of a vaccine for AIDS. Although this vector is typically administered intramuscularly, it would be desirable to induce mucosal immunity by delivery through alternative routes. In this study, the immune response and biodistribution of ADV vectors delivered by different routes were evaluated. ADV vectors expressing human immunodeficiency virus type 1 (HIV-1) Gag, Pol, and Env were delivered intramuscularly or intranasally into mice. Intranasal immunization induced greater HIV-specific immunoglobulin A (IgA) responses in mucosal secretions and sera than in animals with intramuscular injection, which showed stronger systemic cellular and IgG responses. Administration of the vaccine through an intranasal route failed to overcome prior ADV immunity. Animals exposed to ADV prior to vaccination displayed substantially reduced cellular and humoral immune responses to HIV antigens in both groups, though the reduction was greater in animals immunized intranasally. This inhibition was partially overcome by priming with a DNA expression vector expressing HIV-1 Gag, Pol, and Env before boosting with the viral vector. Biodistribution of recombinant adenovirus (rADV) vectors administered intranasally revealed infection of the central nervous system, specifically in the olfactory bulb, possibly via retrograde transport by olfactory neurons in the nasal epithelium, which may limit the utility of this route of delivery of ADV vector-based vaccines.
Restriction of Amino Acid Change in Influenza A Virus H3HA: Comparison of Amino Acid Changes Observed in Nature and In Vitro
Authors: Nakajima Katsuhisa , Nobusawa Eri , Tonegawa Ken , Nakajima Setsuko
Published: August 2003

We introduced 248 single-point amino acid changes into hemagglutinin (HA) protein of the A/Aichi/2/68 (H3N2) strain by a PCR random mutation method. These changes were classified as positive or negative according to their effect on hemadsorption activity. We observed following results. (i) The percentage of surviving amino acid changes on the HA1 domain that did not abrogate hemadsorption activity was calculated to be ca. 44%. In nature, it is estimated to be ca. 39.6%. This difference in surviving amino acid changes on the HA protein between natural isolates and in vitro mutants might be due to the immune pressure against the former. (ii) A total of 26 amino acid changes in the in vitro mutants matched those at which mainstream amino acid changes had occurred in the H3HA1 polypeptide from 1968 to 2000. Of these, 25 were positive. We suggest that the majority of amino acid changes on the HA protein during evolution might be restricted to those that were positive on the HA of A/Aichi/2/68. (iii) We constructed two-point amino acid changes on the HA protein by using positive mutants. These two-point amino acid changes with a random combination did not inhibit hemadsorption activity. It is possible that an accumulation of amino acid change might occur without order. (iv) From the analysis of amino acids participating in mainstream amino acid change, each antigenic site could be further divided into smaller sites. The amino acid substitutions in the gaps between these smaller sites resulted in mostly hemadsorption-negative changes. These gap positions may play an important role in maintaining the function of the HA protein, and therefore amino acid changes are restricted at these locations.
Combinations of Two Capsid Regions Controlling Canine Host Range Determine Canine Transferrin Receptor Binding by Canine and Feline Parvoviruses
Authors: Hueffer Karsten , Govindasamy Lakshman , Agbandje-McKenna Mavis , Parrish Colin R.
Published: August 2003

Feline panleukopenia virus (FPV) and its host range variant, canine parvovirus (CPV), can bind the feline transferrin receptor (TfR), while only CPV binds to the canine TfR. Introducing two CPV-specific changes into FPV (at VP2 residues 93 and 323) endowed that virus with the canine TfR binding property and allowed canine cell infection, although neither change alone altered either property. In CPV the reciprocal changes of VP2 residue 93 or 323 to the FPV sequences individually resulted in modest reductions in infectivity for canine cells. Changing both residues in CPV to the FPV amino acids blocked the canine cell infection, but that virus was still able to bind the canine TfR at low levels. This shows that both CPV-specific changes control canine TfR binding but that binding is not always sufficient to mediate infection.