bluuebunny/crossref_metadata_2025 huggingface dataset:
Involvement of Notch1 Signaling in Neurogenesis in the Subventricular Zone of Normal and Ischemic Rat Brain in Vivo
Authors: Wang XiaoMei , Mao XiaoOu , Xie Lin , Greenberg David A , Jin Kunlin
Published: June 2009

The Notch1 signaling pathway is regarded as one of the main regulators of neural stem cell behavior during development, but its role in the adult brain is less well understood. We found that Notch1 was mainly expressed in doublecortin (DCX)-positive cells corresponding to newborn neurons, whereas the Notch1 ligand, Jagged1, was predominantly expressed in glial fibrillary acidic protein (GFAP)-positive astrocytic cells in the subventricular zone (SVZ) of the normal adult brain. These findings were confirmed by conditional depletion of DCX-positive cells in transgenic mice carrying herpes simplex virus thymidine kinase (HSV-TK) under the control of the DCX promoter. In addition, the activated form of Notch1 (Notch intracellular domain, NICD) and its downstream transcriptional targets, Hes1 and sonic hedgehog (Shh), were also expressed in SVZ cells. Increased activation of Notch1 signaling increased SVZ cell proliferation, whereas inhibiting Notch1 signaling resulted in a reduction of proliferating cells in the SVZ. Levels of NICD, Hes1, and Shh were increased in the SVZ at 4 and 24 h after focal cerebral ischemia. Finally, ischemia-induced cell proliferation in the SVZ was blocked by inhibition of the Notch1 signaling pathway, suggesting that Notch1 signaling may have a key role in normal adult and ischemia-induced neurogenesis.
Integrin α8β1 regulates adhesion, migration and proliferation of human intestinal crypt cells via a predominant RhoA/ROCK‐dependent mechanism
Authors: Benoit Yannick D. , Lussier Carine , Ducharme Pierre‐Alexandre , Sivret Sophie , Schnapp Lynn M. , Basora Nuria , Beaulieu Jean‐François
Published: June 2009

Background. Integrins are transmembrane αβ heterodimer receptors that function as structural and functional bridges between the cytoskeleton and ECM (extracellular matrix) molecules. The RGD (arginine‐glycine‐aspartate tripeptide motif)‐dependent integrin α8β1 has been shown to be involved in various cell functions in neuronal and mesenchymal‐derived cell types. Its role in epithelial cells remains unknown.

Results. Integrin α8β1 was found to be expressed in the crypt cell population of the human intestine but was absent from differentiating and mature epithelial cells of the villus. The function of α8β1 in epithelial crypt cells was investigated at the cellular level using normal HIECs (human intestinal epithelial cells). Specific knockdown of α8 subunit expression using an shRNA (small‐hairpin RNA) approach showed that α8β1 plays important roles in RGD‐dependent cell adhesion, migration and proliferation via a RhoA/ROCK (Rho‐associated kinase)‐dependent mechanism as demonstrated by active RhoA quantification and pharmacological inhibition of ROCK. Moreover, loss of α8β1, through RhoA/ROCK, impairs FA (focal adhesion) complex integrity as demonstrated by faulty vinculin recruitment.

Conclusions. Integrin α8β1 is expressed in epithelial cells. In intestinal crypt cells, α8β1 is closely involved in the regulation of adhesion, migration and cell proliferation via a predominant RhoA/ROCK‐dependent mechanism. These results suggest an important role for this integrin in intestinal crypt cell homoeostasis.
Changes in expression and activity of the secretory pathway Ca2+ ATPase 1 (SPCA1) in A7r5 vascular smooth muscle cells cultured at different glucose concentrations
Authors: Lai Pei , Michelangeli Francesco
Published: June 2009

Diabetes mellitus-related vascular disease is often associated with both a dysregulation of Ca2+ homoeostasis and enhanced secretory activity in VSMCs (vascular smooth muscle cells). Here, we employ a commonly used rat cell line for VSMCs (A7r5 cells) to investigate the effects of glucose on the expression and activity of the SPCA1 (secretory pathway Ca2+-ATPase 1; also known as ATP2C1), which is a P-type Ca2+ pump located in the Golgi apparatus that plays a key role in the secretory pathway. Our results show that mRNA expression levels of SPCA1 are significantly increased in A7r5 cells cultured in high glucose (25.0 mM)-supplemented medium compared with normal glucose (5.55 mM)-supplemented medium. SPCA1 protein expression levels and thapsigargin-insensitive Ca2+-dependent ATPase activity were also consistent with a higher than normal expression level of SPCA1 in high-glucose-cultured A7r5 cells. Analysis of AVP (arginine-vasopressin)-induced cytosolic Ca2+ transients in A7r5 cells (after pre-treatment with thapsigargin) showed faster rise and decay phases in cells grown in high glucose medium compared with cells grown in normal glucose medium, supporting the observation of increased SPCA expression/activity. The significant levels of both Ca2+-ATPase activity and AVP-induced Ca2+ transients, in the presence of thapsigargin, indicate that SPCA must play a significant role in Ca2+ uptake within VSMCs. We therefore propose that, if such increases in SPCA expression and activity also occur in primary VSMCs, this may play a substantial role in the aetiology of diabetes mellitus-associated vascular disease, due to alterations in Ca2+ homoeostasis within the Golgi apparatus.
Adsorption and pinning of multiblock copolymers on chemically heterogeneous patterned surfaces
Authors: Sumithra K. , Brandau M. , Straube E.
Published: June 2009

The results of Monte Carlo simulations carried out on a system of multiblock copolymers having two different types of monomer units adsorbed on checker board surface configurations are presented here. We investigated the adsorption behavior for a series of different chess board square dimensions and also for various block lengths of the copolymer. We find that the specific heat capacity curves for adsorption indicate double peaks showing a two stage pattern recognition of the copolymer on the surface. It is also seen that the transition that happens at lower temperature corresponds to pinning where the junction point of the different blocks gets pinned to the interface between the different surface sites. It is interesting to see that the multiblock copolymers form multiple pinning sites on the boundary between different kinds of surface sites. There exists an intermediate size of the square on the board where the recognition and pinning are most favored and for smaller and larger size of the board and block length, the adsorption proceeds like in homopolymer on homogeneous surfaces. Unlike in the case of the simplest model of diblock copolymer on stripe-patterned surfaces [K. Sumithra and E. Straube, J. Chem. Phys. 125, 154701 (2006)], here the recognition is stronger and the average adsorption energy and the perpendicular component of the radius of gyration show distinct changes corresponding to the two transitions. The conformational properties of the multiblock copolymer near the checkered surface show interesting effects with the perpendicular component showing strong deviations from the standard behavior.
4.3 GHz optical bandwidth light emitting transistor
Authors: Walter G. , Wu C. H. , Then H. W. , Feng M. , Holonyak N.
Published: June 2009

We demonstrate a quantum-well base heterojunction bipolar light emitting transistor (HBLET) operating in the common collector configuration with a 3 dB optical response bandwidth f3 dB of 4.3 GHz. The HBLET has a current gain, β (=|ΔIC/ΔIB|) as high as 30, and can be operated as a three-port device to provide simultaneously an optical and electrical output with gain. The f3 dB of 4.3 GHz corresponds to an effective carrier recombination lifetime of 37 ps, and shows that “fast” spontaneous recombination can be harnessed for high-speed modulation.
Resonant Raman study of phonons in high-quality colloidal CdTe nanoparticles
Authors: Dzhagan V. , Valakh M. Ya. , Kolny-Olesiak J. , Lokteva I. , Zahn D. R. T.
Published: June 2009

We report on a resonant Raman scattering study of colloidal CdTe nanoparticles (NPs). The perfect crystallinity and narrow size distribution of the NPs allowed at least three additional spectral features near the main longitudinal optical phonon peak to be resolved at 50 K. We assign these features to scattering by transverse (145 cm−1) and surface (159 cm−1) optical phonon modes as well as to a combined optical-acoustic phonon mode(s) (180–200 cm−1). Both the transverse and combined optical-acoustic phonon modes were not reported previously for CdTe NPs.
Effects of quantum tunneling in metal nanogap on surface-enhanced Raman scattering
Authors: Mao Li , Li Zhipeng , Wu Biao , Xu Hongxing
Published: June 2009

The quantum tunneling effects between two silver plates are studied using the time dependent density functional theory. Our results show that the tunneling depends mainly on the separation and the initial local field of the interstice between plates. The smaller separation and larger local field, the easier the electrons tunnels through the interstice. Our numerical calculation shows that when the separation is smaller than 0.6 nm the quantum tunneling dramatically reduces the enhancing ability of interstice between nanoparticles.
NAADP-mediated Ca2+signaling via type 1 ryanodine receptor in T cells revealed by a synthetic NAADP antagonist
Authors: Dammermann Werner , Zhang Bo , Nebel Merle , Cordiglieri Chiara , Odoardi Francesca , Kirchberger Tanja , Kawakami Naoto , Dowden James , Schmid Frederike , Dornmair Klaus , Hohenegger Martin , Flügel Alexander , Guse Andreas H. , Potter Barry V. L.
Published: June 2009

The nucleotide NAADP was recently discovered as a second messenger involved in the initiation and propagation of Ca2+signaling in lymphoma T cells, but its impact on primary T cell function is still unknown. An optimized, synthetic, small molecule inhibitor of NAADP action, termed BZ194, was designed and synthesized. BZ194 neither interfered with Ca2+mobilization byd-myo-inositol 1,4,5-trisphosphate or cyclic ADP-ribose nor with capacitative Ca2+entry. BZ194 specifically and effectively blocked NAADP-stimulated [3H]ryanodine binding to the purified type 1 ryanodine receptor. Further, in intact T cells, Ca2+mobilization evoked by NAADP or by formation of the immunological synapse between primary effector T cells and astrocytes was inhibited by BZ194. Downstream events of Ca2+mobilization, such as nuclear translocation of “nuclear factor of activated T cells” (NFAT), T cell receptor-driven interleukin-2 production, and proliferation in antigen-experienced CD4+effector T cells, were attenuated by the NAADP antagonist. Taken together, specific inhibition of the NAADP signaling pathway constitutes a way to specifically and effectively modulate T-cell activation and has potential in the therapy of autoimmune diseases.
Unbiased RNA–protein interaction screen by quantitative proteomics
Authors: Butter Falk , Scheibe Marion , Mörl Mario , Mann Matthias
Published: June 2009

Mass spectrometry (MS)-based quantitative interaction proteomics has successfully elucidated specific protein–protein, DNA–protein, and small molecule–protein interactions. Here, we developed a gel-free, sensitive, and scalable technology that addresses the important area of RNA–protein interactions. Using aptamer-tagged RNA as bait, we captured RNA-interacting proteins from stable isotope labeling by amino acids in cell culture (SILAC)-labeled mammalian cell extracts and analyzed them by high-resolution, quantitative MS. Binders specific to the RNA sequence were distinguished from background by their isotope ratios between bait and control. We demonstrated the approach by retrieving known and novel interaction partners for the HuR interaction motif, H4 stem loop, “zipcode” sequence, tRNA, and a bioinformatically-predicted RNA fold in DGCR-8/Pasha mRNA. In all experiments we unambiguously identified known interaction partners by a single affinity purification step. The 5′ region of the mRNA of DGCR-8/Pasha, a component of the microprocessor complex, specifically interacts with components of the translational machinery, suggesting that it contains an internal ribosome entry site.
Microfabricated tissue gauges to measure and manipulate forces from 3D microtissues
Authors: Legant Wesley R. , Pathak Amit , Yang Michael T. , Deshpande Vikram S. , McMeeking Robert M. , Chen Christopher S.
Published: June 2009

Physical forces generated by cells drive morphologic changes during development and can feedback to regulate cellular phenotypes. Because these phenomena typically occur within a 3-dimensional (3D) matrix in vivo, we used microelectromechanical systems (MEMS) technology to generate arrays of microtissues consisting of cells encapsulated within 3D micropatterned matrices. Microcantilevers were used to simultaneously constrain the remodeling of a collagen gel and to report forces generated during this process. By concurrently measuring forces and observing matrix remodeling at cellular length scales, we report an initial correlation and later decoupling between cellular contractile forces and changes in tissue morphology. Independently varying the mechanical stiffness of the cantilevers and collagen matrix revealed that cellular forces increased with boundary or matrix rigidity whereas levels of cytoskeletal and extracellular matrix (ECM) proteins correlated with levels of mechanical stress. By mapping these relationships between cellular and matrix mechanics, cellular forces, and protein expression onto a bio-chemo-mechanical model of microtissue contractility, we demonstrate how intratissue gradients of mechanical stress can emerge from collective cellular contractility and finally, how such gradients can be used to engineer protein composition and organization within a 3D tissue. Together, these findings highlight a complex and dynamic relationship between cellular forces, ECM remodeling, and cellular phenotype and describe a system to study and apply this relationship within engineered 3D microtissues.
The glucanosyltransferase Gas1 functions in transcriptional silencing
Authors: Koch Melissa R. , Pillus Lorraine
Published: June 2009

Transcriptional silencing is a crucial process that is mediated through chromatin structure. The histone deacetylase Sir2 silences genomic regions that include telomeres, ribosomal DNA (rDNA) and the cryptic mating-type loci. Here, we report an unsuspected role for the enzyme Gas1 in locus-specific transcriptional silencing.
GAS1
encodes a β-1,3-glucanosyltransferase previously characterized for its role in cell wall biogenesis. In
gas1
mutants, telomeric silencing is defective and rDNA silencing is enhanced. We show that the catalytic activity of Gas1 is required for normal silencing, and that Gas1's role in silencing is distinct from its role in cell wall biogenesis. Established hallmarks of silent chromatin, such as Sir2 and Sir3 binding, H4K16 deacetylation, and H3K56 deacetylation, appear unaffected in
gas1
mutants. Thus, another event required for telomeric silencing must be influenced by
GAS1
. Because the catalytic activity of Gas1 is required for telomeric silencing, Gas1 localizes to the nuclear periphery, and Gas1 and Sir2 physically interact, we propose a model in which carbohydrate modification of chromatin components provides a new regulatory element that may be critical for chromatin function but which is virtually unexplored in the current landscape of chromatin analysis.
Direct observation of an ensemble of stable collapsed states in the mechanical folding of ubiquitin
Authors: Garcia-Manyes Sergi , Dougan Lorna , Badilla Carmen L. , Brujić Jasna , Fernández Julio M.
Published: June 2009

Statistical theories of protein folding have long predicted plausible mechanisms for reducing the vast conformational space through distinct ensembles of structures. However, these predictions have remained untested by bulk techniques, because the conformational diversity of folding molecules has been experimentally unapproachable. Owing to recent advances in single molecule force-clamp spectroscopy, we are now able to probe the structure and dynamics of the small protein ubiquitin by measuring its length and mechanical stability during each stage of folding. Here, we discover that upon hydrophobic collapse, the protein rapidly selects a subset of minimum energy structures that are mechanically weak and essential precursors of the native fold. From this much reduced ensemble, the native state is acquired through a barrier-limited transition. Our results support the validity of statistical mechanics models in describing the folding of a small protein on biological timescales.
Dynamic features of gene expression control by small regulatory RNAs
Authors: Mitarai Namiko , Benjamin Julie-Anna M. , Krishna Sandeep , Semsey Szabolcs , Csiszovszki Zsolt , Massé Eric , Sneppen Kim
Published: June 2009

Small regulatory RNAs (sRNAs) in eukaryotes and bacteria play an important role in the regulation of gene expression either by binding to regulatory proteins or directly to target mRNAs. Two of the best-characterized bacterial sRNAs, Spot42 and RyhB, form a complementary pair with the ribosome binding region of their target mRNAs, thereby inhibiting translation or promoting mRNA degradation. To investigate the steady-state and dynamic potential of such sRNAs, we examine the 2 key parameters characterizing sRNA regulation: the capacity to overexpress the sRNA relative to its target mRNA and the speed at which the target mRNA is irreversibly inactivated. We demonstrate different methods to determine these 2 key parameters, for Spot42 and RyhB, which combine biochemical and genetic experiments with computational analysis. We have developed a mathematical model that describes the functional properties of sRNAs with various characteristic parameters. We observed that Spot42 and RyhB function in distinctive parameter regimes, which result in divergent mechanisms.
Real-time molecular monitoring of chemical environment in obligate anaerobes during oxygen adaptive response
Authors: Holman Hoi-Ying N. , Wozei Eleanor , Lin Zhang , Comolli Luis R. , Ball David A. , Borglin Sharon , Fields Matthew W. , Hazen Terry C. , Downing Kenneth H.
Published: June 2009

Determining the transient chemical properties of the intracellular environment can elucidate the paths through which a biological system adapts to changes in its environment, for example, the mechanisms that enable some obligate anaerobic bacteria to survive a sudden exposure to oxygen. Here we used high-resolution Fourier transform infrared (FTIR) spectromicroscopy to continuously follow cellular chemistry within living obligate anaerobes by monitoring hydrogen bond structures in their cellular water. We observed a sequence of well orchestrated molecular events that correspond to changes in cellular processes in those cells that survive, but only accumulation of radicals in those that do not. We thereby can interpret the adaptive response in terms of transient intracellular chemistry and link it to oxygen stress and survival. This ability to monitor chemical changes at the molecular level can yield important insights into a wide range of adaptive responses.
Activation of tumor cell integrin α
v
β
3
controls angiogenesis and metastatic growth in the brain
Authors: Lorger Mihaela , Krueger Joseph S. , O'Neal Melissa , Staflin Karin , Felding-Habermann Brunhilde
Published: June 2009

The incidence of brain metastasis is rising and poses a severe clinical problem, as we lack effective therapies and knowledge of mechanisms that control metastatic growth in the brain. Here we demonstrate a crucial role for high-affinity tumor cell integrin α
v
β
3
in brain metastatic growth and recruitment of blood vessels. Although α
v
β
3
is frequently up-regulated in primary brain tumors and metastatic lesions of brain homing cancers, we show that it is the α
v
β
3
activation state that is critical for brain lesion growth. Activated, but not non-activated, tumor cell α
v
β
3
supports efficient brain metastatic growth through continuous up-regulation of vascular endothelial growth factor (VEGF) protein under normoxic conditions. In metastatic brain lesions carrying activated α
v
β
3
, VEGF expression is controlled at the post-transcriptional level and involves phosphorylation and inhibition of translational respressor 4E-binding protein (4E-BP1). In contrast, tumor cells with non-activated α
v
β
3
depend on hypoxia for VEGF induction, resulting in reduced angiogenesis, tumor cell apoptosis, and inefficient intracranial growth. Importantly, the microenvironment critically influences the effects that activated tumor cell α
v
β
3
exerts on tumor cell growth. Although it strongly promoted intracranial growth, the activation state of the receptor did not influence tumor growth in the mammary fat pad as a primary site. Thus, we identified a mechanism by which metastatic cells thrive in the brain microenvironment and use the high-affinity form of an adhesion receptor to grow and secure host support for proliferation. Targeting this molecular mechanism could prove valuable for the inhibition of brain metastasis.
Sodium-calcium exchanger complexed with GM1 ganglioside in nuclear membrane transfers calcium from nucleoplasm to endoplasmic reticulum
Authors: Wu Gusheng , Xie Xin , Lu Zi-Hua , Ledeen Robert W.
Published: June 2009

The inner membrane of the nuclear envelope (NE) was previously shown to contain a Na/Ca exchanger (NCX) tightly linked to GM1 ganglioside that mediates transfer of nucleoplasmic Ca
2+
to the NE lumen and constitutes a cytoprotective mechanism. This transfer was initially observed with isolated nuclei and is now demonstrated in living cells in relation to subcellular Ca
2+
dynamics. Four cell lines with varying expression of NCX and GM1 in the NE were transfected with cameleon-fluorescent Ca
2+
indicators genetically targeted to NE/endoplasmic reticulum (ER) and nucleoplasm to monitor [Ca
2+
]
ne/er
and [Ca
2+
]
n
respectively. Cytosolic Ca
2+
([Ca
2+
]
cyt
) was indicated with fura-2. Thapsigargin caused progressive loss of [Ca
2+
]
ne/er
, which was rapidly replaced on addition of extrinsic Ca
2+
to those cells containing fully functional NCX/GM1: differentiated NG108–15 and C6 cells. Reduced elevation of [Ca
2+
]
ne/er
following thapsigargin depletion occurred in cells containing little or no GM1 in the NE: undifferentiated NG108–15 and NG-CR72 cells. No change in [Ca
2+
]
ne/er
due to applied Ca
2+
was seen in Jurkat cells, which entirely lack NCX. Ca
2+
entry to NE/ER was also blocked by KB-R7943, inhibitor of NCX. [Ca
2+
]
n
and [Ca
2+
]
cyt
were elevated independent of [Ca
2+
]
ne/er
and remained in approximate equilibrium with each other. Ca
2+
rise in the ER originated in the NE region and extended to the entire ER network. These results indicate the nuclear NCX/GM1 complex acts to gate Ca
2+
transfer from cytosol to ER, an alternate route to the sarcoplasmic/endoplasmic reticulum calcium ATPase pump. They also suggest a possible contributory mechanism for independent regulation of nuclear Ca
2+
.
Temporal lenses for attosecond and femtosecond electron pulses
Authors: Hilbert Shawn A. , Uiterwaal Cornelis , Barwick Brett , Batelaan Herman , Zewail Ahmed H.
Published: June 2009

Here, we describe the “temporal lens” concept that can be used for the focus and magnification of ultrashort electron packets in the time domain. The temporal lenses are created by appropriately synthesizing optical pulses that interact with electrons through the ponderomotive force. With such an arrangement, a temporal lens equation with a form identical to that of conventional light optics is derived. The analog of ray diagrams, but for electrons, are constructed to help the visualization of the process of compressing electron packets. It is shown that such temporal lenses not only compensate for electron pulse broadening due to velocity dispersion but also allow compression of the packets to durations much shorter than their initial widths. With these capabilities, ultrafast electron diffraction and microscopy can be extended to new domains,and, just as importantly, electron pulses can be delivered directly on an ultrafast techniques target specimen.
Antenatal ultrasound and postnatal autopsy findings in terminations after 12 weeks’ gestation due to fetal abnormality: population-based study in western norway, 1988–2002
Authors: Parkar A. P. , Olsen Ø. E. , Maartmann-Moe H. , Daltveit A. K. , Gjelland K. , Rosendahl K.
Published: June 2009

Background: Ultrasound screening has been part of antenatal care for several decades, and warrants high expertise to meet the criteria for a worthwhile screening program. In particular, the rate of false positives should be low.

Purpose: To examine time trends of pregnancy terminations for fetal abnormality after 12 weeks’ gestation, and to assess the agreement between antenatal ultrasound and post-termination autopsy findings for the main pathologies leading to termination.

Material and Methods: During the period 1988 to 2002, 198 pregnancies were terminated for fetal abnormality after 12 weeks’ gestation. We reviewed the case notes for those 151 who were autopsied (male/female/undetermined ;= ;91/56/4). Annual rates of live births and stillbirths were retrieved from the Medical Birth Registry of Norway.

Results: Antenatal ultrasound provided a correct diagnosis of the major abnormality in 149/151 cases (99%), based on post-termination autopsy findings. The annual rate of terminations after 12 weeks’ gestation varied between 0.6 and 3.4 (mean 1.8) per 1000 live births, with a trend toward higher rates over the study period ( P=0.001, chi-square test for linear-by-linear association).

Conclusion: The specificity of antenatal ultrasound for major abnormalities was high, as compared to postnatal autopsy findings. The mean annual rates of termination after 12 weeks’ gestation tended to increase over the 14-year study period.
HUMMR, a hypoxia- and HIF-1α–inducible protein, alters mitochondrial distribution and transport
Authors: Li Yan , Lim Seung , Hoffman David , Aspenstrom Pontus , Federoff Howard J. , Rempe David A.
Published: June 2009

Mitochondrial transport is critical for maintenance of normal neuronal function. Here, we identify a novel mitochondria protein, hypoxia up-regulated mitochondrial movement regulator (HUMMR), which is expressed in neurons and is markedly induced by hypoxia-inducible factor 1 α (HIF-1α). Interestingly, HUMMR interacts with Miro-1 and Miro-2, mitochondrial proteins that are critical for mediating mitochondrial transport. Interestingly, knockdown of HUMMR or HIF-1 function in neurons exposed to hypoxia markedly reduces mitochondrial content in axons. Because mitochondrial transport and distribution are inextricably linked, the impact of reduced HUMMR function on the direction of mitochondrial transport was also explored. Loss of HUMMR function in hypoxia diminished the percentage of motile mitochondria moving in the anterograde direction and enhanced the percentage moving in the retrograde direction. Thus, HUMMR, a novel mitochondrial protein induced by HIF-1 and hypoxia, biases mitochondria transport in the anterograde direction. These findings have broad implications for maintenance of neuronal viability and function during physiological and pathological states.
Impact of Gene Copy Number Variation on Anesthesia in Drosophila melanogaster
Authors: Alone Debasmita P. , Rodriguez Jason C. , Noland Cameron L. , Nash Howard A.
Published: June 2009

Background

Chromosomal deletions and duplications, which result in halving or doubling of copy number in a block of genes, are an important source of variation between individuals. Phenotypic effects of copy number variation are commonly observed, but effects on sensitivity to volatile anesthetics have not been assessed in any organism.

Methods

The potency with which halothane depresses the righting reflex of fruit flies was measured in congenic Drosophila strains, each of which was heterozygous for a deletion of average size 400 kb. Over 200 strains were examined, thereby scanning approximately half of the fly genome.

Results

Although the vast majority of deletion heterozygotes were indistinguishable from the control, eight had significantly altered sensitivity to halothane. Genetic tests supported the hypothesis that the change in anesthetic sensitivity was the result of reduction in copy number and not adventitious mutations in the strains. Among the eight outliers, the difference in halothane potency ranged from a 25% increase to a 15% decrease. Changes of similar magnitude but distinctive patterns were found when these lines were tested with enflurane, isoflurane, and sevoflurane.

Conclusions

Variation in gene copy number has a significant impact on anesthetic sensitivity in Drosophila melanogaster. The level of transcription of a few genes must thus be limiting for a normal response to volatiles. Coupling between gene copy and gene expression is universal, and the components of the fly's nervous system are highly conserved; therefore, this work provides a rationale for investigating the clinical impact of copy number variation.
Evolution of Changes in Upper Airway Collapsibility during Slow Induction of Anesthesia with Propofol
Authors: Hillman David R. , Walsh Jennifer H. , Maddison Kathleen J. , Platt Peter R. , Kirkness Jason P. , Noffsinger William J. , Eastwood Peter R.
Published: June 2009

Background

Upper airway collapsibility is known to increase under anesthesia. This study assessed how this increase in collapsibility evolves during slow Propofol induction and how it relates to anesthesia-induced changes in upper airway muscle activity and conscious state.

Methods

Nine healthy volunteers were studied. Anesthesia was induced with Propofol in a step-wise manner (effect-site concentration steps of 0.5 microg x ml(-1) from 0 to 3 microg x ml(-1) and thereafter to 4 microg x ml(-1) and 6 microg x ml(-1) [target-controlled infusion]). Airway patency was maintained with continuous positive airway pressure. Pharyngeal collapsibility was assessed at each concentration by measuring critical pressure. Intramuscular genioglossus electromyogram and anesthetic depth (bispectral index score) were monitored throughout. Loss of consciousness was defined as failure to respond to loud verbal command.

Results

Loss of consciousness occurred at varying Propofol effect-site concentrations between 1.5 and 4.0 microg x ml(-1). Initially genioglossus electromyographic activity was sustained with increases in Propofol concentration, increasing in some individuals. At or approaching loss of consciousness, it decreased, often abruptly, to minimal values with an accompanying increase in critical pressure. In most subjects, bispectral index score decreased alinearly with increasing Propofol concentration with greatest rate of change coinciding with loss of consciousness.

Conclusions

Slow stepwise induction of Propofol anesthesia is associated with an alinear increase in upper airway collapsibility. Disproportionate decreases in genioglossus electromyogram activity and increases in pharyngeal critical closing pressure were observed proximate to loss of consciousness, suggesting that particular vulnerability exists after transition from conscious to unconscious sedation. Such changes may have parallels with upper airway behavior at sleep onset.
Coapplication of Lidocaine and the Permanently Charged Sodium Channel Blocker QX-314 Produces a Long-lasting Nociceptive Blockade in Rodents
Authors: Binshtok Alexander M. , Gerner Peter , Oh Seog Bae , Puopolo Michelino , Suzuki Suzuko , Roberson David P. , Herbert Teri , Wang Chi-Fei , Kim Donghoon , Chung Gehoon , Mitani Aya A. , Wang Ging Kuo , Bean Bruce P. , Woolf Clifford J.
Published: June 2009

Background

Nociceptive-selective local anesthesia is produced by entry of the permanently charged lidocaine-derivative QX-314 into nociceptors when coadministered with capsaicin, a transient receptor potential vanilloid 1 (TRPV1) channel agonist. However, the pain evoked by capsaicin before establishment of the QX-314-mediated block would limit clinical utility. Because TRPV1 channels are also activated by lidocaine, the authors tested whether lidocaine can substitute for capsaicin to introduce QX-314 into nociceptors through TRPV1 channels and produce selective analgesia.

Methods

Lidocaine (0.5% [17.5 mM], 1% [35 mM], and 2% [70 mM]) alone, QX-314 (0.2% [5.8 mM]) alone, and a combination of the two were injected subcutaneously and adjacent to the sciatic nerve in rats and mice. Mechanical and thermal responsiveness were measured, as was motor block.

Results

Coapplication of 0.2% QX-314 with lidocaine prolonged the nociceptive block relative to lidocaine alone, an effect attenuated in TRPV1 knockout mice. The 0.2% QX-314 alone had no effect when injected intraplantary or perineurally, and it produced only weak short-lasting inhibition of the cutaneous trunci muscle reflex. Perisciatic nerve injection of lidocaine with QX-314 produced a differential nociceptive block much longer than the transient motor block, lasting 2 h (for 1% lidocaine) to 9 h (2% lidocaine). Triple application of lidocaine, QX-314, and capsaicin further increased the duration of the differential block.

Conclusions

Coapplication of lidocaine and its quaternary derivative QX-314 produces a long-lasting, predominantly nociceptor-selective block, likely by facilitating QX-314 entry through TRPV1 channels. Delivery of QX-314 into nociceptors by using lidocaine instead of capsaicin produces sustained regional analgesia without nocifensive behavior.
Nefopam and Alfentanil Additively Reduce the Shivering Threshold in Humans whereas Nefopam and Clonidine Do Not
Authors: Alfonsi Pascal , Passard Andrea , Gaude–Joindreau Valérie , Guignard Bruno , Sessler Daniel I. , Chauvin Marcel
Published: June 2009

Background

Induction of therapeutic hypothermia is often complicated by shivering. Nefopam reduces the shivering threshold with minimal side effects. Consequently, nefopam is an attractive component for induction of therapeutic hypothermia. However, nefopam alone is insufficient; it will thus need to be combined with another drug. Clonidine and alfentanil each reduce the shivering threshold. This study, therefore, tested the hypothesis that nefopam, combined either with clonidine or alfentanil, synergistically reduces the shivering threshold.

Methods

For each combination, ten volunteers were studied on 4 days. Combination 1: (1) control (no drug); (2) nefopam (100 ng/ml); (3) clonidine (2.5 microg/kg); and (4) nefopam plus clonidine (100 ng/ml and 2.5 microg/kg, respectively). Combination 2: (1) control (no drug); (2) nefopam (100 ng/ml); (3) alfentanil (150 ng/ml); and (4) nefopam plus alfentanil (100 ng/ml and 150 ng/ml, respectively). Lactated Ringer's solution (approximately 4 degrees C) was infused to decrease core temperature. Mean skin temperature was maintained at 31 degrees C. The core temperature that increased oxygen consumption to more than 25% of baseline identified the shivering threshold.

Results

With nefopam and clonidine, the shivering thresholds were significantly lower than on the control day. The shivering threshold decreased significantly less than would be expected on the basis of the individual effects of each drug (P = 0.034). In contrast, the interaction between nefopam and alfentanil on shivering was additive, meaning that the combination reduced the shivering threshold as much as would be expected by the individual effect of each drug.

Conclusions

Nefopam and alfentanil additively reduce the shivering threshold, but nefopam and clonidine do not.
Evolution and conservation in human parechovirus genomes
Authors: Williams Çiğdem H. , Panayiotou Maria , Girling Gareth D. , Peard Curtis I. , Oikarinen Sami , Hyöty Heikki , Stanway Glyn
Published: June 2009

Human parechoviruses (HPeVs) are frequent pathogens with a seroprevalance of over 90 % in adults. Recent studies on these viruses have increased the number of HPeV types to eight. Here we analyse the complete genome of one clinical isolate, PicoBank/HPeV1/a, and VP1 and 3D protein sequences of PicoBank/HPeV6/a, isolated from the same individual 13 months later. PicoBank/HPeV1/a is closely related to other recent HPeV1 isolates but is distinct from the HPeV1 Harris prototype isolated 50 years ago. The availability of an increasing number of HPeV sequences has allowed a detailed analysis of these viruses. The results add weight to the observations that recombination plays a role in the generation of HPeV diversity. An important finding is the presence of unexpected conservation of codons utilized in part of the 3D-encoding region, some of which can be explained by the presence of a phylogenetically conserved predicted secondary structure domain. This suggests that in addition to thecis-acting replication element, RNA secondary structure domains in coding regions play a key role in picornavirus replication.
Acidic residues in the membrane-proximal stalk region of vaccinia virus protein B5 are required for glycosaminoglycan-mediated disruption of the extracellular enveloped virus outer membrane
Authors: Roberts Kim L. , Breiman Adrien , Carter Gemma C. , Ewles Helen A. , Hollinshead Michael , Law Mansun , Smith Geoffrey L.
Published: June 2009

The extracellular enveloped virus (EEV) form of vaccinia virus (VACV) is surrounded by two lipid envelopes. This presents a topological problem for virus entry into cells, because a classical fusion event would only release a virion surrounded by a single envelope into the cell. Recently, we described a mechanism in which the EEV outer membrane is disrupted following interaction with glycosaminoglycans (GAGs) on the cell surface and thus allowing fusion of the inner membrane with the plasma membrane and penetration of a naked core into the cytosol. Here we show that both the B5 and A34 viral glycoproteins are required for this process. A34 is required to recruit B5 into the EEV membrane and B5 acts as a molecular switch to control EEV membrane rupture upon exposure to GAGs. Analysis of VACV strains expressing mutated B5 proteins demonstrated that the acidic stalk region between the transmembrane anchor sequence and the fourth short consensus repeat of B5 are critical for GAG-induced membrane rupture. Furthermore, the interaction between B5 and A34 can be disrupted by the addition of polyanions (GAGs) and polycations, but only the former induce membrane rupture. Based on these data we propose a revised model for EEV entry.
Inhibition of epidermal growth factor receptor (EGFR) expression by human cytomegalovirus correlates with an increase in the expression and binding of Wilms' Tumour 1 protein to the EGFR promoter
Authors: Jafferji Insiya , Bain Mark , King Christine , Sinclair John H.
Published: June 2009

Infection with human cytomegalovirus (HCMV) modulates the expression of a number of cellular receptors and is known to inhibit expression of the epidermal growth factor receptor (EGFR), a cell surface receptor that can promote cell proliferation through a cascade of intracellular signalling events. We have examined the mechanisms by which HCMV mediates downregulation of EGFR expression and show that virus infection results in the profound upregulation of Wilms' Tumour 1 (WT1) protein, a transcription factor associated with the negative regulation of a number of growth factors and growth factor receptors, including EGFR. Moreover, chromatin immunoprecipitation experiments also show that HCMV infection results in increased binding of WT1 to the EGFR promoter. Finally, we show that depleting the cell of WT1 using small interfering RNA abrogates virus-mediated downregulation of EGFR. Taken together, our observations suggest that HCMV-mediated repression of EGFR expression results from a virus-mediated increase in cellular WT1, a known pleiotropic regulator of mitogenesis, apoptosis and differentiation.
Differential expression of microRNAs in Marek's disease virus-transformed T-lymphoma cell lines
Authors: Yao Yongxiu , Zhao Yuguang , Smith Lorraine P. , Lawrie Charles H. , Saunders Nigel J. , Watson Michael , Nair Venugopal
Published: June 2009

MicroRNAs (miRNAs) are increasingly recognized to play crucial roles in regulation of gene expression in different biological events, including many sporadic forms of cancer. However, despite the involvement of several viruses in inducing cancer, only a limited number of studies have been carried out to examine the miRNA expression signatures in virus-induced neoplasia, particularly in herpesvirus-induced tumours where virus-encoded miRNAs also contribute significantly to the miRNome of the tumour cell. Marek's disease (MD) is a naturally occurring, rapid-onset CD4+T-cell lymphoma of poultry, induced by the highly contagious Marek's disease virus (MDV). High levels of expression of virus-encoded miRNAs and altered expression of several host-encoded miRNAs were demonstrated in the MDV-transformed lymphoblastoid cell line MSB-1. In order to identify the miRNA expression signature specific to MDV-transformed cells, we examined the global miRNA expression profiles in seven distinct MDV-transformed cell lines by microarray analysis. This study revealed that, in addition to the high levels of MDV-encoded miRNAs, these MD tumour-derived lymphoblastoid cell lines showed altered expression of several host-encoded miRNAs. Comparison of the miRNA expression profiles of these cell lines with the MDV-negative, retrovirus-transformed AVOL-1 cell line showed that miR-150 and miR-223 are downregulated irrespective of the viral aetiology, whereas downregulation of miR-155 was specific for MDV-transformed tumour cells. Thus, increased expression of MDV-encoded miRNAs with specific downregulation of miR-155 can be considered as unique expression signatures for MD tumour cells. Analysis of the functional targets of these miRNAs would contribute to the understanding of the molecular pathways of MD oncogenicity.
Morphological changes in human neural cells following tick-borne encephalitis virus infection
Authors: Růžek Daniel , Vancová Marie , Tesařová Martina , Ahantarig Arunee , Kopecký Jan , Grubhoffer Libor
Published: June 2009

Tick-borne encephalitis (TBE) is one of the leading and most dangerous human viral neuroinfections in Europe and north-eastern Asia. The clinical manifestations include asymptomatic infections, fevers and debilitating encephalitis that might progress into chronic disease or fatal infection. To understand TBE pathology further in host nervous systems, three human neural cell lines, neuroblastoma, medulloblastoma and glioblastoma, were infected with TBE virus (TBEV). The susceptibility and virus-mediated cytopathic effect, including ultrastructural and apoptotic changes of the cells, were examined. All the neural cell lines tested were susceptible to TBEV infection. Interestingly, the neural cells produced about 100- to 10 000-fold higher virus titres than the conventional cell lines of extraneural origin, indicating the highly susceptible nature of neural cells to TBEV infection. The infection of medulloblastoma and glioblastoma cells was associated with a number of major morphological changes, including proliferation of membranes of the rough endoplasmic reticulum and extensive rearrangement of cytoskeletal structures. The TBEV-infected cells exhibited either necrotic or apoptotic morphological features. We observed ultrastructural apoptotic signs (condensation, margination and fragmentation of chromatin) and other alterations, such as vacuolation of the cytoplasm, dilatation of the endoplasmic reticulum cisternae and shrinkage of cells, accompanied by a high density of the cytoplasm. On the other hand, infected neuroblastoma cells did not exhibit proliferation of membranous structures. The virions were present in both the endoplasmic reticulum and the cytoplasm. Cells were dying preferentially by necrotic mechanisms rather than apoptosis. The neuropathological significance of these observations is discussed.
Characterization of a virion occlusion-defective Autographa californica multiple nucleopolyhedrovirus mutant lacking the p26, p10 and p74 genes
Authors: Wang Lihua , Salem Tamer Z. , Campbell Dean J. , Turney Colin M. , Kumar C. M. Senthil , Cheng Xiao-Wen
Published: June 2009

Nucleopolyhedroviruses (NPVs), family Baculoviridae, are insect-specific viruses with the potential to control insect pests in agriculture and forestry. NPVs are occluded in polyhedral occlusion bodies. Polyhedra protect virions from inactivation in the environment as well as assisting virions in horizontal transmission in the insect population. The process of virion occlusion in the polyhedra is undefined and the genes that regulate the virion occlusion process have not been well investigated yet. An Autographa californica multiple nucleopolyhedrovirus (AcMNPV) mutant (AcDef) that has a 2136 bp DNA deletion, including p26, p10 and p74 genes, has been isolated. No virions were detected in the polyhedra of AcDef. Restoration of all the missing sequences into AcDef led to proper virion occlusion. Individual gene deletion of either p10 or p26 could not abolish virion occlusion in the polyhedra of AcMNPV, but p10 deletion reduced virion occlusion efficiency more than threefold compared with the wild-type AcMNPV. Previous studies by other research groups on deletion of AcMNPV gene p74 suggested that p74 is a per os infectivity factor, and deletion of the p74 gene did not eliminate virion occlusion. Collectively, the three genes (p26, p10 and p74) may act in concert to regulate the virion occlusion process. Therefore, p26, p10 and p74 are all required for proper virion occlusion in the polyhedra of AcMNPV.
Location of antigenic sites recognized by monoclonal antibodies in the influenza A virus nucleoprotein molecule
Authors: Varich Natalia L. , Kochergin-Nikitsky Konstantin S. , Usachev Evgeny V. , Usacheva Olga V. , Prilipov Alexei G. , Webster Robert G. , Kaverin Nikolai V.
Published: June 2009

The locations of amino acid positions relevant to antigenic variation in the nucleoprotein (NP) of influenza virus are not conclusively known. We analysed the antigenic structure of influenza A virus NP by introducing site-specific mutations at amino acid positions presumed to be relevant for the differentiation of strain differences by anti-NP monoclonal antibodies. Mutant proteins were expressed in a prokaryotic system and analysed by performing ELISA with monoclonal antibodies. Four amino acid residues were found to determine four different antibody-binding sites. When mapped in a 3D X-ray model of NP, the four antigenically relevant amino acid positions were found to be located in separate physical sites of the NP molecule.
RNA triphosphatase and guanylyl transferase activities are associated with the RNA polymerase protein L of rinderpest virus
Authors: Gopinath M. , Shaila M. S.
Published: June 2009

Rinderpest virus (RPV) large (L) protein is an integral part of the ribonucleoprotein (RNP) complex of the virus that is responsible for transcription and replication of the genome. Previously, we have shown that recombinant L protein coexpressed along with P protein (as the L–P complex) catalyses the synthesis of all viral mRNAsin vitroand the abundance of mRNAs follows a gradient of polarity, similar to the occurrencein vivo. In the present work, we demonstrate that the viral mRNAs synthesizedin vitroby the recombinant L or purified RNP are capped and methylated at the N7guanine position. RNP from the purified virions, as well as recombinant L protein, shows RNA triphosphatase (RTPase) and guanylyl transferase (GT) activities. L protein present in the RNP complex catalyses the removal ofγ-phosphate from triphosphate-ended 25 nt RNA generatedin vitrorepresenting the viral N-terminal mRNA 5′ sequence. The L protein forms a covalent enzyme–guanylate intermediate with the GMP moiety of GTP, whose formation is inhibited by the addition of pyrophosphate; thus, it exhibits characteristics of cellular GTs. The covalent bond between the enzyme and nucleotide is acid labile and alkali stable, indicating the presence of phosphoamide linkage. The C-terminal region (aa 1717–2183) of RPV L protein alone exhibits the first step of GT activity needed to form a covalent complex with GMP, though it lacks the ability to transfer GMP to substrate RNA. Here, we describe the biochemical characterization of the newly found RTPase/GT activity of L protein.