bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of Nonstructural Protein NS2A in Flavivirus Assembly
Authors: Leung Jason Y. , Pijlman Gorben P. , Kondratieva Natasha , Hyde Jennifer , Mackenzie Jason M. , Khromykh Alexander A.
Published: March 2008

Flavivirus nonstructural (NS) proteins are involved in RNA replication and modulation of the host antiviral response; however, evidence is mounting that some NS proteins also have essential roles in virus assembly. Kunjin virus (KUN) NS2A is a small, hydrophobic, transmembrane protein that is part of the replication complex and inhibits interferon induction. Previously, we have shown that an isoleucine (I)-to-asparagine (N) substitution at position 59 of the NS2A protein blocked the production of secreted virus particles in cells electroporated with viral RNA carrying this mutation. We now show that prolonged incubation of mutant KUN NS2A-I59N replicon RNA, in an inducible BHK-derived packaging cell line (expressing KUN structural proteins C, prM, and E), generated escape mutants that rescued the secretion of infectious virus-like particles. Sequencing identified three groups of revertants that included (i) reversions to wild-type, hydrophobic Ile, (ii) pseudorevertants to more hydrophobic residues (Ser, Thr, and Tyr) at codon 59, and (iii) pseudorevertants retaining Asn at NS2A codon 59 but containing a compensatory mutation (Thr-to-Pro) at NS2A codon 149. Engineering hydrophobic residues at NS2A position 59 or the compensatory T149P mutation into NS2A-I59N replicon RNA restored the assembly of secreted virus-like particles in packaging cells. T149P mutation also rescued virus production when introduced into the full-length KUN RNA containing an NS2A-I59N mutation. Immunofluorescence and electron microscopy analyses of NS2A-I59N replicon-expressing cells showed a distinct lack of virus-induced membranes normally present in cells expressing wild-type replicon RNA. The compensatory mutation NS2A-T149P restored the induction of membrane structures to a level similar to those observed during wild-type replication. The results further confirm the role of NS2A in virus assembly, demonstrate the importance of hydrophobic residues at codon 59 in this process, implicate the involvement of NS2A in the biogenesis of virus-induced membranes, and suggest a vital role for the virus-induced membranes in virus assembly.
Role for the L1-52/55K Protein in the Serotype Specificity of Adenovirus DNA Packaging
Authors: Wohl Beverly P. , Hearing Patrick
Published: March 2008

The packaging of adenovirus (Ad) DNA into virions is dependent upon
cis
-acting sequences and
trans
-acting proteins. We studied the involvement of Ad packaging proteins in the serotype specificity of packaging. Both Ad5 and Ad17 IVa2 and L4-22K proteins complemented the growth of Ad5 IVa2 and L4-22K mutant viruses, respectively. In contrast, the Ad5 L1-52/55K protein complemented an Ad5 L1-52/55K mutant virus, but the Ad17 L1-52/55K protein did not. The analysis of chimeric proteins demonstrated that the N-terminal half of the Ad5 L1-52/55K protein mediated this function. Finally, we demonstrate that the L4-33K and L4-22K proteins have distinct functions during infection.
Systemic and Brain Macrophage Infections in Relation to the Development of Simian Immunodeficiency Virus Encephalitis
Authors: Bissel Stephanie J. , Wang Guoji , Bonneh-Barkay Dafna , Starkey Adam , Trichel Anita M. , Murphey-Corb Michael , Wiley Clayton A.
Published: March 2008

The brains of individuals with lentiviral-associated encephalitis contain an abundance of infected and activated macrophages. It has been hypothesized that encephalitis develops when increased numbers of infected monocytes traffic into the central nervous system (CNS) during the end stages of immunosuppression. The relationships between the infection of brain and systemic macrophages and circulating monocytes and the development of lentiviral encephalitis are unknown. We longitudinally examined the extent of monocyte/macrophage infection in blood and lymph nodes of pigtailed macaques that did or did not develop simian immunodeficiency virus encephalitis (SIVE). Compared to levels in macaques that did not develop SIVE, more ex vivo virus production was detected from monocyte-derived macrophages and nonadherent peripheral blood mononuclear cells (PBMCs) from macaques that did develop SIVE. Prior to death, there was an increase in the number of circulating PBMCs following a rise in cerebrospinal fluid viral load in macaques that did develop SIVE but not in nonencephalitic macaques. At necropsy, macaques with SIVE had more infected macrophages in peripheral organs, with the exception of lymph nodes. T cells and NK cells with cytotoxic potential were more abundant in brains with encephalitis; however, T-cell and NK-cell infiltration in SIVE and human immunodeficiency virus encephalitis was more modest than that observed in classical acute herpes simplex virus encephalitis. These findings support the hypothesis that inherent differences in host systemic and CNS monocyte/macrophage viral production are associated with the development of encephalitis.
Dimerization of the Human Papillomavirus Type 16 E2 N Terminus Results in DNA Looping within the Upstream Regulatory Region
Authors: Hernandez-Ramon Elena E. , Burns Julie E. , Zhang Wenke , Walker Hannah F. , Allen Stephanie , Antson Alfred A. , Maitland Norman J.
Published: March 2008

Papillomavirus E2 proteins play a central role in regulating viral gene expression and replication. DNA-binding activity is associated with the C-terminal domain of E2, which forms a stable dimer, while the N-terminal domain is responsible for E2's replication and transactivation functions. The crystal structure of the latter domain revealed a second dimerization interface on E2 which may be responsible for DNA loop formation in the regulatory region of the human papillomavirus (HPV) genome. We investigated the biological significance of the N-terminal dimerization by introducing single amino acid substitutions into the dimerization interface. As expected, these substitutions did not influence the C-terminal dimerization and DNA-binding functions of E2. However, the mutations led to reduced transactivation of a synthetic E2-responsive reporter gene, while HPV DNA replication was unaffected. The effect of the mutations on DNA looping was visualized by atomic force microscopy. While wild-type E2 was able to generate DNA loops, all three mutant E2 proteins were defective in this ability. Our results suggest that N-terminal dimerization plays a role in E2-mediated transactivation, probably via DNA looping, a common mechanism for remote regulation of gene transcription.
Live Visualization of Herpes Simplex Virus Type 1 Compartment Dynamics
Authors: de Oliveira Anna Paula , Glauser Daniel L. , Laimbacher Andrea S. , Strasser Regina , Schraner Elisabeth M. , Wild Peter , Ziegler Urs , Breakefield Xandra O. , Ackermann Mathias , Fraefel Cornel
Published: March 2008

We have constructed a recombinant herpes simplex virus type 1 (HSV-1) that simultaneously encodes selected structural proteins from all three virion compartments—capsid, tegument, and envelope—fused with autofluorescent proteins. This triple-fluorescent recombinant, rHSV-RYC, was replication competent, albeit with delayed kinetics, incorporated the fusion proteins into all three virion compartments, and was comparable to wild-type HSV-1 at the ultrastructural level. The VP26 capsid fusion protein (monomeric red fluorescent protein [mRFP]-VP26) was first observed throughout the nucleus and later accumulated in viral replication compartments. In the course of infection, mRFP-VP26 formed small foci in the periphery of the replication compartments that expanded and coalesced over time into much larger foci. The envelope glycoprotein H (gH) fusion protein (enhanced yellow fluorescent protein [EYFP]-gH) was first observed accumulating in a vesicular pattern in the cytoplasm and was then incorporated primarily into the nuclear membrane. The VP16 tegument fusion protein (VP16-enhanced cyan fluorescent protein [ECFP]) was first observed in a diffuse nuclear pattern and then accumulated in viral replication compartments. In addition, it also formed small foci in the periphery of the replication compartments which, however, did not colocalize with the small mRFP-VP26 foci. Later, VP16-ECFP was redistributed out of the nucleus into the cytoplasm, where it accumulated in vesicular foci and in perinuclear clusters reminiscent of the Golgi apparatus. Late in infection, mRFP-VP26, EYFP-gH, and VP16-ECFP were found colocalizing in dots at the plasma membrane, possibly representing mature progeny virus. In summary, this study provides new insights into the dynamics of compartmentalization and interaction among capsid, tegument, and envelope proteins. Similar strategies can also be applied to assess other dynamic events in the virus life cycle, such as entry and trafficking.
Transcriptome Analysis of Infection of the Archaeon
Sulfolobus solfataricus
with
Sulfolobus
Turreted Icosahedral Virus
Authors: Ortmann Alice C. , Brumfield Susan K. , Walther Jasper , McInnerney Kathleen , Brouns Stan J. J. , van de Werken Harmen J. G. , Bothner Brian , Douglas Trevor , van de Oost John , Young Mark J.
Published: March 2008

Microarray analysis of infection by
Sulfolobus t
urreted
i
cosahedral
v
irus (STIV) revealed insights into the timing and extent of virus transcription, as well as differential regulation of host genes. Using a microarray containing genes from both the host and the virus, the infection cycle of STIV was studied. Following infection of
Sulfolobus solfataricus
strain 2-2-12 with STIV, transcription of virus genes was first detected at 8 h postinfection (p.i.), with a peak at 24 h p.i. Lysis of cells was first detected at 32 h p.i. There was little temporal control of the transcription of virus genes, although the three open reading frames on the noncoding strand were transcribed later in the infection process. During the infection, 177 host genes were determined to be differentially expressed, with 124 genes up-regulated and 53 genes down-regulated. The up-regulated genes were dominated by genes associated with DNA replication and repair and those of unknown function, while the down-regulated genes, mostly detected at 32 h p.i., were associated with energy production and metabolism. Examination of infected cells by transmission electron microscopy revealed alterations in cell ultrastructure consistent with the microarray analysis. The observed patterns of transcription suggest that up-regulated genes are likely used by the virus to reprogram the cell for virus replication, while the down-regulated genes reflect the imminent lysis of the cells.
Transmission of Simian Immunodeficiency Virus Carrying Multiple Cytotoxic T-Lymphocyte Escape Mutations with Diminished Replicative Ability Can Result in AIDS Progression in Rhesus Macaques
Authors: Seki Sayuri , Kawada Miki , Takeda Akiko , Igarashi Hiroko , Sata Tetsutaro , Matano Tetsuro
Published: March 2008

Cytotoxic T-lymphocyte (CTL) responses frequently select for immunodeficiency virus mutations that result in escape from CTL recognition with viral fitness costs. The replication in vivo of such viruses carrying not single but multiple escape mutations in the absence of the CTL pressure has remained undetermined. Here, we have examined the replication of simian immunodeficiency virus (SIV) with five
gag
mutations selected in a macaque possessing the major histocompatibility complex haplotype
90
-
120
-
Ia
after its transmission into
90
-
120
-
Ia
-negative macaques. Our results showed that even such a “crippled” SIV infection can result in persistent viral replication, multiple reversions, and AIDS progression.
Magnitude and Phenotype of Cellular Immune Responses Elicited by Recombinant Adenovirus Vectors and Heterologous Prime-Boost Regimens in Rhesus Monkeys
Authors: Liu Jinyan , Ewald Bonnie A. , Lynch Diana M. , Denholtz Matthew , Abbink Peter , Lemckert Angelique A. C. , Carville Angela , Mansfield Keith G. , Havenga Menzo J. , Goudsmit Jaap , Barouch Dan H.
Published: March 2008

Recombinant adenovirus serotype 5 (rAd5) vaccine vectors for human immunodeficiency virus type 1 (HIV-1) and other pathogens have been shown to elicit antigen-specific cellular immune responses. Rare serotype rAd vectors have also been constructed to circumvent preexisting anti-Ad5 immunity and to facilitate the development of novel heterologous rAd prime-boost regimens. Here we show that rAd5, rAd26, and rAd48 vectors elicit qualitatively distinct phenotypes of cellular immune responses in rhesus monkeys and can be combined as potent heterologous prime-boost vaccine regimens. While rAd5-Gag induced primarily gamma interferon-positive (IFN-γ
+
) and IFN-γ
+
/tumor necrosis factor alpha
+
(TNF-α
+
) T-lymphocyte responses, rAd26-Gag and rAd48-Gag induced higher proportions of interleukin-2
+
(IL-2
+
) and polyfunctional IFN-γ
+
/TNF-α
+
/IL-2
+
T-lymphocyte responses. Priming with the rare serotype rAd vectors proved remarkably effective for subsequent boosting with rAd5 vectors. These data demonstrate that the rare serotype rAd vectors elicited T-lymphocyte responses that were phenotypically distinct from those elicited by rAd5 vectors and suggest the functional relevance of polyfunctional CD8
+
and CD4
+
T-lymphocyte responses. Moreover, qualitative differences in cellular immune responses may prove critical in determining the overall potency of heterologous rAd prime-boost regimens.
SYMMETRIES OF FRACTAL TILINGS
Authors: PALAGALLO JUDITH , SALCEDO MARIA
Published: March 2008

Self-similar tilings of the plane can be generated by an iterative replacement property using squares and triangles. We show tilings with tiles that are topological disks and others whose tiles are connected but have an interior that is disconnected. In each example we note the symmetries of the individual tiles and describe how each tiling exhibits a property common to crystallographic tilings.
SPECTRA OF SELF-SIMILAR LAPLACIANS ON THE SIERPINSKI GASKET WITH TWISTS
Authors: BLASIAK ANNA , STRICHARTZ ROBERT S. , UGURCAN BARIS EVREN
Published: March 2008

We study the spectra of a two-parameter family of self-similar Laplacians on the Sierpinski gasket (SG) with twists. By this we mean that instead of the usual IFS that yields SG as its invariant set, we compose each mapping with a reflection to obtain a new IFS that still has SG as its invariant set, but changes the definition of self-similarity. Using recent results of Cucuringu and Strichartz, we are able to approximate the spectra of these Laplacians by two different methods. To each Laplacian we associate a self-similar embedding of SG into the plane, and we present experimental evidence that the method of outer approximation, recently introduced by Berry, Goff and Strichartz, when applied to this embedding, yields the spectrum of the Laplacian (up to a constant multiple).
ROTATION NUMBERS OF INVARIANT MANIFOLDS AROUND UNSTABLE PERIODIC ORBITS FOR THE DIAMAGNETIC KEPLER PROBLEM
Authors: WU ZUO-BING
Published: March 2008

In this paper, a method to construct topological template in terms of symbolic dynamics for the diamagnetic Kepler problem is proposed. To confirm the topological template, rotation numbers of invariant manifolds around unstable periodic orbits in a phase space are taken as an object of comparison. The rotation numbers are determined from the definition and connected with symbolic sequences encoding the periodic orbits in a reduced Poincaré section. Only symbolic codes with inverse ordering in the forward mapping can contribute to the rotation of invariant manifolds around the periodic orbits. By using symbolic ordering, the reduced Poincaré section is constricted along stable manifolds and a topological template, which preserves the ordering of forward sequences and can be used to extract the rotation numbers, is established. The rotation numbers computed from the topological template are the same as those computed from their original definition.
BIPEDICLE FLAP RECONSTRUCTION FOR COMPLEX LONGITUDINAL DORSAL TISSUE DEFECTS OF THE DIGITS
Authors: Fujiwara Masao , Ito Ran , Fukamizu Hidekazu
Published: March 2008

Achieving coverage after digital injury is crucial, because simple skin defects can expose essential structures such as tendons or bones. This is particularly true on the dorsal surfaces of the digits, where the skin provides the only protection for the tendons. However, longitudinal skin defects of the digit have not been specifically identified in the literature and there have been few reports focusing on longitudinal dorsal skin defects. Here we report on the use of a bipedicle flap for reconstruction of complex longitudinal dorsal tissue defects of the digits, including those associated with tendon or bone damage.
A PROSPECTIVE PLAN TO MINIMISE MEDIAN NERVE RELATED COMPLICATIONS ASSOCIATED WITH OPERATIVELY TREATED DISTAL RADIUS FRACTURES
Authors: Henry Mark , Stutz Christopher
Published: March 2008

Loss of median nerve function or a neuropathic pain syndrome may occur in around 20% of distal radius fractures if post-traumatic oedema in the carpal canal generates excessive pressure on the median nerve. No method currently exists to reliably distinguish which patients may benefit from a concomitant carpal tunnel release. This case series details the results of following a prospective plan designed to minimise median nerve related complications associated with distal radius fractures by measuring Semmes-Weinstein monofilament scores in 374 radius fracture patients who underwent surgical stabilisation. One hundred and sixty-nine patients with the clinical symptoms of median nerve compression, a decrement in monofilament score of grade 1 (out of 5) compared to the contralateral side or at least 4.31 g underwent concomitant carpal tunnel release. The remaining 205 patients did not have carpal tunnel release. There were no cases of neuropathic pain or loss of median nerve function.
LATE REDUCTION FOR A CHRONIC TRANS-SCAPHOID PERILUNATE DISLOCATION IN A SKELETALLY IMMATURE 15-YEAR-OLD BOY: A CASE REPORT
Authors: Siwicka Karolina , Horii Emiko
Published: March 2008

Fracture dislocation of the wrist is a rare injury in adolescents, and therefore it is easily ignored at the initial treatment. Once ignored, an alternative treatment such as proximal row carpectomy is indicated, but surgical outcome is not as good as that of an early reduction. We have experienced a chronic case of fracture dislocation in a 15-year-old, skeletally immature boy and treated it by scaphoid osteotomy, associated with bone grafting, screw fixation and ligament repair. The patient had no difficulties in daily activities nine years post-operatively, however the X-ray showed slight deformity of the scaphoid. Even for a chronic case, late reduction with ligamentous repair should be considered in adolescents.
Wavelet Processing of Palladium Foil Images from Atomic Force Microscopy (AFM) Measurements
Authors: Lowrie Chris , Earles Susan
Published: March 2008

Discrete wavelet image processing techniques are applied to AFM images of Palladium for improving image quality. In this paper Discrete Wavelet Transforms (DWT) are used to process AFM images to minimize the noise introduced by AFM instrumentation errors. The smallest of mechanical and electrical errors from the AFM introduces noise into the images. We will use the DWT to reduce noise and to compress the image size. Wavelet denoising can remove gaussian noise as well as nonlinearly generated instrumentation noise, and wavelet compression greatly reduces the number of pixels required to store a large number of images.
Multi-sensory, Data Fusion Detection System for Improved Situational Awareness
Authors: Rose-Pehrsson Susan L. , Minor Christian , Johnson Kevin , Owrutsky Jeff , Wales Stephen , Steinhurst Daniel , Gottuk Daniel
Published: March 2008

A multi-sensory approach is being used to develop new detection capabilities for improved damage assessment and real-time situational awareness. The detection system combines surveillance camera video images with selected spectral and acoustic signatures and image recognition technologies to provide a broad range of situational awareness. Various spectral and acoustic signatures, new video imaging techniques, and image recognition methods have been investigated and integrated into a multi-sensory prototype system. The prototype system is able to detect event signatures within the volume of a space (i.e., a "volume sensor") rather than relying on spot-type fire detectors. Two prototype systems were built and assessed in full-scale testing aboard the ex-USS Shadwell side-by-side with two commercial video image fire detection systems and several spot-type fire detection systems. Tests included a wide range of fire and nuisance sources, plus flooding and pipe rupture scenarios under actual shipboard background conditions. The prototype systems are shown to outperform the commercial fire detection systems for flaming and smoldering fires with a high level of nuisance immunity. In addition, they successfully detected the pipe ruptures and flooding scenarios. The system can be adapted for homeland security.
Metal-Insulator-Gap-Insulator-Semiconductor structure for Biological Sensors
Authors: Hashimoto Hideaki , Hirokane Takaaki , Kanzaki Daisuke , Urabe Shinichi , Morita Mizuho
Published: March 2008

A metal-insulator-gap-insulator-semiconductor sensing device has been characterized in different pH solutions and with different single strand DNA solutions by capacitance-voltage measurements. The capacitance-voltage curves show the difference from pH and the difference from DNA base by hysteresis and flat band voltage shift due to mobile ionic charge in the solution. As the pH decreases, the flat band voltage shift increases in the pH range of 2.7 to 7.0. The hysteresis in the capacitance-voltage curves shows the influence of ionic charge in the solutions and the change of the sensing surface condition. The difference of the flat band voltage shift in the capacitance-voltage curves is related to the mobile ionic charge in the solutions due to pH or DNA molecules.
Electrochemical Transducers - A New Approach to Ultrasound Sensor Design
Authors: Shamsi Atif , Bae Sang-Eun , Majkic Goran , Brankovic Stanko R.
Published: March 2008

We present a study investigating the electrochemical interface as a capacitive transducer for ultrasound sensor applications. The concept is demonstrated using a nanoporous Au layer as a sensing electrode. The sensing electrode was produced by dealloying of the Ag0.8Au0.2 bulk electrode yielding an Au layer with extremely high surface/volume ratio. This electrode configuration was tested against the state of the art piezoelectric sensor subjected to electrolyte excitation with ultrasound waves having 40 kHz frequency and 4000 Pa amplitude. Our results demonstrate that nanoporous Au layer can be used as a capacitive transducer for sensing dynamic changes of pressure in electrolytes (ultrasound sensing). The sensitivity of this electrode can be readily adjusted by increasing the thickness of the dealloyed electrode layer. The transducer equation is developed and its practical relevance is discussed for electrochemical ultrasound sensor design.
Evaluation of Parylene as Protection Layer for Capacitive Micromachined Ultrasonic Transducers
Authors: Jeanne Edgard , Meynier Cyril , Terry Jonathan , Roy Mathieu , Haworth Les , Alquier Daniel
Published: March 2008

Due to their enhanced performance, capacitive Micromachined Ultrasonic Transducers (cMUTs) are on the way to replacing standard piezoelectric ceramics technology in ultrasound medical imaging. The cMUT device requires the implementation of a final protection layer in order to avoid electrode degradation and achieve biocompatibility. Parylene C is a material of choice for such applications as it can be deposited in exceptionally conformal and chemically inert thin films. In this work, the influence of 1µm thick protective coating of Parylene film on cMUT performance and reliability is studied through simulations and dynamic characterization. Data obtained by laser Doppler vibrometry show a good agreement between simulations and experiments with a slight increase of the collapse voltage. Device ageing provides evidence of the necessity and the effectiveness of Parylene as a protective layer. Finally, this work reveals that cMUT resonant frequency can be easily tuned by controlling the deposited Parylene film thickness.
Corrosion Protection of Alternative Coatings on High-Strength 4130 Steel
Authors: Yoon Yuhchae , Petry Leanne , Hansen D. C.
Published: March 2008

The electrochemical behavior of high-strength 4130 steel with alternative coating systems as well as current cadmium plating and chromate conversion coatings have been investigated using anodic and cathodic polarization, electrochemical impedance and ion chromatography techniques. During anodic polarization, Cd-chromate conversion coated, aluminum-chrome, SermeTel T, and Alseal basecoat exhibited sacrificial coating protection ability while ion vapor deposited-aluminum, SermeTel W, SermeTel W basecoat and SermeTel T basecoat exhibited barrier coating protection. The cathodic polarization data shows that most of the coatings tested exhibit a cathodic corrosion resistance except for the Alseal basecoat. Results of the electrochemical impedance measurements indicate that SermeTel W exhibited barrier protection properties while the remaining coatings exhibited conductive properties. Visual inspection of the IVD-Al and SermeTel W coatings indicated they were intact after 6 days immersion in 5 wt% NaCl. The remaining coatings exhibited surface damage.
FGF-16 is released from neonatal cardiac myocytes and alters growth-related signaling: a possible role in postnatal development
Authors: Lu Shun Yan , Sontag David P. , Detillieux Karen A. , Cattini Peter A.
Published: March 2008

FGF-16 has been reported to be preferentially expressed in the adult rat heart. We have investigated the expression of FGF-16 in the perinatal and postnatal heart and its functional significance in neonatal rat cardiac myocytes. FGF-16 mRNA accumulation was observed by quantitative RT-PCR between neonatal days 1 and 7, with this increased expression persisting into adulthood. FGF-2 has been shown to increase neonatal rat cardiac myocyte proliferative potential via PKC activation. Gene array analysis revealed that FGF-16 inhibited the upregulation by FGF-2 of cell cycle promoting genes including cyclin F and Ki67. Furthermore, the CDK4/6 inhibitor gene Arf/INK4A was upregulated with the combination of FGF-16 and FGF-2 but not with either factor on its own. The effect on Ki67 was validated by protein immunodetection, which also showed that FGF-16 significantly decreased FGF-2-induced Ki67 labeling of cardiac myocytes, although it alone had no effect on Ki67 labeling. Inhibition of p38 MAPK potentiated cardiac myocyte proliferation induced by FGF-2 but did not alter the inhibitory action of FGF-16. Receptor binding assay showed that FGF-16 can compete with FGF-2 for binding sites including FGF receptor 1. FGF-16 had no effect on activated p38, ERK1/2, or JNK/SAPK after FGF-2 treatment. However, FGF-16 inhibited PKC-α and PKC-ε activation induced by FGF-2 and, importantly, IGF-1. Collectively, these data suggest that expression and release of FGF-16 in the neonatal myocardium interfere with cardiac myocyte proliferative potential by altering the local signaling environment via modulation of PKC activation and cell cycle-related gene expression.
TRPV1-mediated protection against endotoxin-induced hypotension and mortality in rats
Authors: Wang Youping , Novotný Martin , Quaiserová-Mocko Veronika , Swain Greg M. , Wang Donna H.
Published: March 2008

This study was designed to test the hypothesis that the transient receptor potential vanilloid type 1 (TRPV1) channel, expressed primarily in sensory nerves, and substance P (SP), released by sensory nerves, play a protective role against lipopolysaccharide (LPS)-induced hypotension. LPS (10 mg/kg iv) elicited tachycardia and hypotension in anesthetized male Wistar rats, which peaked at 10 min and gradually recovered 1 h after the injection. Blockade of TRPV1 with its selective antagonist capsazepine (CAPZ, 3 mg/kg iv) impaired recovery given that the fall in mean arterial pressure (MAP) was greater 1 h after CAPZ plus LPS injections compared with LPS injection alone (45 ± 5 vs. 25 ± 4 mmHg, P < 0.05). Blockade of the neurokinin 1 (NK1) receptor with its selective antagonists RP-67580 (5 mg/kg iv) or L-733,060 (4 mg/kg iv) prevented recovery, considering that falls in MAP were not different 1 h after injections of NK1 antagonists plus LPS from their peak decreases (66 ± 9 vs. 74 ± 5 mmHg or 60 ± 7 vs. 69 ± 3 mmHg, respectively, P > 0.05). LPS increased plasma SP, norepinephrine (NE), and epinephrine (Epi) levels compared with vehicles, and the increases in plasma SP, NE, and Epi were significantly inhibited by CAPZ or RP-67580. The survival rate at 24 or 48 h after LPS injection (20 mg/kg ip) was lower in conscious rats pretreated with CAPZ or RP-67580 compared with rats treated with LPS alone ( P < 0.05). Thus our results show that the TRPV1, possibly via triggering release of SP which activates the NK1 and stimulates the sympathetic axis, plays a protective role against endotoxin-induced hypotension and mortality, suggesting that TRPV1 receptors are essential in protecting vital organ perfusion and survival during the endotoxic condition.
Forskolin Induces NMDA Receptor-Dependent Potentiation at a Central Synapse in the Leech
Authors: Grey Kathryn B. , Burrell Brian D.
Published: March 2008

In vertebrate hippocampal neurons, application of forskolin (an adenylyl cyclase activator) and rolipram (a phosphodiesterase inhibitor) is an effective technique for inducing chemical long-term potentiation (cLTP) that is N-methyl-d-aspartate (NMDA) receptor (NMDAR)-dependent. However, it is not known whether forskolin induces a similar potentiation in invertebrate synapses. Therefore, we examined whether forskolin plus rolipram treatment could induce potentiation at a known glutamatergic synapse in the leech ( Hirudo sp.), specifically between the pressure (P) mechanosensory and anterior pagoda (AP) neurons. Perfusion of isolated ganglia with forskolin (50 μM) in conjunction with rolipram (0.1 μM) in Mg2+-free saline significantly potentiated the P-to-AP excitatory postsynaptic potential. Application of 2-amino-5-phosphonovaleric acid (APV, 100 μM), a competitive NMDAR antagonist, blocked the potentiation, indicating P-to-AP potentiation is NMDAR-dependent. Potentiation was blocked by injection of bis-( o-aminophenoxy)- N,N,N′, N′-tetraacetic acid (BAPTA, 1 mM) into the postsynaptic cell, but not by BAPTA injection into the presynaptic neuron, indicating a requirement for postsynaptic elevation of intracellular Ca2+. Application of db-cAMP mimicked the potentiating effects of forskolin, and Rp-cAMP, an inhibitor of protein kinase A, blocked forskolin-induced potentiation. Potentiation was also blocked by autocamtide-2-related inhibitory peptide (AIP), indicating a requirement for activation of Ca2+/calmodulin-dependent kinase II (CaMKII). Finally, potentiation was blocked by botulinum toxin, suggesting that trafficking of glutamate receptors also plays a role in this form of synaptic plasticity. These experiments demonstrate that techniques used to induce cLTP in vertebrate synapses also induce NMDAR-dependent potentiation in the leech CNS and that many of the cellular processes that mediate LTP are conserved between vertebrate and invertebrate phyla.
Body-Tilt and Visual Verticality Perception During Multiple Cycles of Roll Rotation
Authors: Vingerhoets R.A.A. , Medendorp W. P. , Van Gisbergen J.A.M.
Published: March 2008

To assess the effects of degrading canal cues for dynamic spatial orientation in human observers, we tested how judgments about visual-line orientation in space (subjective visual vertical task, SVV) and estimates of instantaneous body tilt (subjective body-tilt task, SBT) develop in the course of three cycles of constant-velocity roll rotation. These abilities were tested across the entire tilt range in separate experiments. For comparison, we also obtained SVV data during static roll tilt. We found that as tilt increased, dynamic SVV responses became strongly biased toward the head pole of the body axis (A-effect), as if body tilt was underestimated. However, on entering the range of near-inverse tilts, SVV responses adopted a bimodal pattern, alternating between A-effects (biased toward head-pole) and E-effects (biased toward feet-pole). Apart from an onset effect, this tilt-dependent pattern of systematic SVV errors repeated itself in subsequent rotation cycles with little sign of worsening performance. Static SVV responses were qualitatively similar and consistent with previous reports but showed smaller A-effects. By contrast, dynamic SBT errors were small and unimodal, indicating that errors in visual-verticality estimates were not caused by errors in body-tilt estimation. We discuss these results in terms of predictions from a canal-otolith interaction model extended with a leaky integrator and an egocentric bias mechanism. We conclude that the egocentric-bias mechanism becomes more manifest during constant velocity roll-rotation and that perceptual errors due to incorrect disambiguation of the otolith signal are small despite the decay of canal signals.
Neurotensin Enhances GABAergic Activity in Rat Hippocampus CA1 Region by Modulating L-Type Calcium Channels
Authors: Li Shanshan , Geiger Jonathan D. , Lei Saobo
Published: March 2008

Neurotensin (NT) is a tridecapeptide that interacts with three NT receptors; NTS1, NTS2, and NTS3. Although NT has been reported to modulate GABAergic activity in the brain, the underlying cellular and molecular mechanisms of NT are elusive. Here, we examined the effects of NT on GABAergic transmission and the involved cellular and signaling mechanisms of NT in the hippocampus. Application of NT dose-dependently increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) recorded from CA1 pyramidal neurons with no effects on the amplitude of sIPSCs. NT did not change either the frequency or the amplitude of miniature (m)IPSCs recorded in the presence of tetrodotoxin. Triple immunofluorescent staining of recorded interneurons demonstrated the expression of NTS1 on GABAergic interneurons. NT increased the action potential firing rate but decreased the afterhyperpolarization (AHP) amplitude in identified CA1 interneurons. Application of L-type calcium channel blockers (nimodipine and nifedipine) abolished NT-induced increases in action potential firing rate and sIPSC frequency and reduction in AHP amplitude, suggesting that the effects of NT are mediated by interaction with L-type Ca2+channels. NT-induced increase in sIPSC frequency was blocked by application of the specific NTS1 antagonist SR48692, the phospholipase C (PLC) inhibitor U73122, the IP3receptor antagonist 2-APB, and the protein kinase C inhibitor GF109203X, suggesting that NT increases γ-aminobutyric acid release via a PLC pathway. Our results provide a cellular mechanism by which NT controls GABAergic neuronal activity in hippocampus.
Artificial Dirt: Microfluidic Substrates for Nematode Neurobiology and Behavior
Authors: Lockery S. R. , Lawton K. J. , Doll J. C. , Faumont S. , Coulthard S. M. , Thiele T. R. , Chronis N. , McCormick K. E. , Goodman M. B. , Pruitt B. L.
Published: March 2008

With a nervous system of only 302 neurons, the free-living nematode Caenorhabditis elegans is a powerful experimental organism for neurobiology. However, the laboratory substrate commonly used in C. elegans research, a planar agarose surface, fails to reflect the complexity of this organism's natural environment, complicates stimulus delivery, and is incompatible with high-resolution optophysiology experiments. Here we present a new class of microfluidic devices for C. elegans neurobiology and behavior: agarose-free, micron-scale chambers and channels that allow the animals to crawl as they would on agarose. One such device mimics a moist soil matrix and facilitates rapid delivery of fluid-borne stimuli. A second device consists of sinusoidal channels that can be used to regulate the waveform and trajectory of crawling worms. Both devices are thin and transparent, rendering them compatible with high-resolution microscope objectives for neuronal imaging and optical recording. Together, the new devices are likely to accelerate studies of the neuronal basis of behavior in C. elegans.
Hypoxia-Inducible Factor-1 Target Genes as Indicators of Tumor Vessel Response to Vascular Endothelial Growth Factor Inhibition
Authors: Dang Duyen T. , Chun Sang Y. , Burkitt Kyunghee , Abe Masako , Chen Shaowei , Havre Pamela , Mabjeesh Nicola J. , Heath Elisabeth I. , Vogelzang Nicholas J. , Cruz-Correa Marcia , Blayney Douglas W. , Ensminger William D. , Croix Brad St. , Dang Nam H. , Dang Long H.
Published: March 2008

Antiangiogenic therapy improves survival in patients with advanced stage cancers. Currently, there are no reliable predictors or markers for tumor vessel response to antiangiogenic therapy. To model effective antiangiogenic therapy, we disrupted the VEGF gene in three representative cancer cell lines. HCT116 xenografts had low proportions of endothelial tubes covered by pericytes that stained with α-smooth muscle actin (SMA) antibody. Upon disruption of VEGF, HCT116VEGF−/− xenografts had significantly decreased tumor microvessel perfusion compared with their parental counterparts. Furthermore, HCT116VEGF−/− xenografts mounted a tumor-reactive response to hypoxia, characterized by the induction of hypoxia-inducible factor-1 (HIF-1) target genes. One highly induced protein was DPP4, a measurable serum protein that has well-described roles in cancer progression. In contrast, LS174T and MKN45 tumor xenografts had high proportion of endothelial tubes that were covered by SMA+ pericytes. Upon disruption of VEGF, LS174TVEGF−/− and MKN45VEGF−/− xenografts maintained tumor microvessel perfusion. As such, there were no changes in intratumoral hypoxia or HIF-1α induction. Together, these data show that the extent of tumor vessel response to angiogenic inhibition could be correlated with (a) the preexisting coverage of tumor endothelial tubes with SMA+ pericytes and (b) differential tumor induction of HIF-1 target genes. The data further show that DPP4 is a novel marker of HIF-1 induction. Altogether, these preclinical findings suggest novel clinical trials for predicting and monitoring tumor vessel responses to antiangiogenic therapy. [Cancer Res 2008;68(6):1872–80]
Adjuvant Therapy with Small Hairpin RNA Interference Prevents Non–Small Cell Lung Cancer Metastasis Development in Mice
Authors: Bulk Etmar , Hascher Antje , Liersch Ruediger , Mesters Rolf M. , Diederichs Sven , Sargin Bülent , Gerke Volker , Hotfilder Marc , Vormoor Josef , Berdel Wolfgang E. , Serve Hubert , Müller-Tidow Carsten
Published: March 2008

Development of distant metastasis is the major reason for cancer-related deaths worldwide. Adjuvant therapy approaches after local therapies are most effective when specific targets are inhibited. Recently, we identified S100P overexpression as a strong predictor for metastasis development in early-stage non–small cell lung cancer (NSCLC) patients. Here, we show that S100P overexpression increased angiogenesis in and metastasis formation from s.c. xenotransplants of NSCLC cells. Plasmid-derived short hairpin RNAs (shRNA) were developed as specific adjuvant therapy. I.v. injected shRNA against S100P significantly decreased S100P protein expression in xenograft tumors and inhibited tumor angiogenesis in vivo. Metastasis formation 8 weeks after primary tumor resection was significantly reduced. Lung metastases developed in 31% of mice treated with S100P-targeting shRNAs compared with 64% in control shRNA–treated mice (P < 0.05). These findings suggest that RNA interference–based therapy approaches can be highly effective in the adjuvant setting. [Cancer Res 2008;68(6):1896–904]
Chemopreventive Effect of Silymarin on Liver Pathology in HBV X Protein Transgenic Mice
Authors: Wu Yi-Fang , Fu Shu-Ling , Kao Cheng-Heng , Yang Chu-Wen , Lin Chao-Hsiung , Hsu Ming-Ta , Tsai Ting-Fen
Published: March 2008

There are currently limited therapeutic regimens available for effective treatment of hepatocellular carcinoma (HCC). Silymarin is a naturally derived polyphenolic antioxidant with hepatoprotective properties and is very widely used in clinical application; however, effect of silymarin on spontaneous HCC has not been studied. Silymarin was evaluated for its efficacy against spontaneous carcinogenesis using the HBV X protein (HBx) transgenic model. Silymarin was p.o. given to the HBx transgenic mice from 4 to 6 weeks of age. Our data indicated that silymarin has therapeutic effects on the early stages of liver damage, reversing fatty changes and recovering liver histopathology in a dose-dependent manner. To study the chemopreventive effects on the later stages of carcinogenesis, the mice at 13 months were split into a precancerous group and a group with significant liver carcinogenesis. After silymarin was given to the precancerous mice from 13 to 16 months of age, in contrast to an 80% incidence of HCC development in the untreated transgenic mice, no HCC was detected in any of these mice. Nonetheless, small hyperplastic nodules were detected in 86% of these precancerous mice. In the second group with notable HCC, silymarin was unable to block cancer progression. Although silymarin did not affect HBx expression, intracellular reactive oxygen species levels were decreased, cell proliferation was stimulated, and hepatocyte ultrastructure was found to significantly recover. In conclusion, silymarin exerts beneficial effects on the early stages of liver pathogenesis, preventing and delaying liver carcinogenesis. This drug should be considered as a potential chemopreventive agent for HBV-related hepatocarcinogenesis. [Cancer Res 2008;68(6):2033–42]
Tumor Cell Dependence on Ran-GTP–Directed Mitosis
Authors: Xia Fang , Lee Connie W. , Altieri Dario C.
Published: March 2008

Deregulated cell division is a hallmark of cancer, but whether tumor cells become dependent on specific mitotic mechanisms is not known. Here, we show that the small GTPase Ran, a regulator of mitotic spindle formation, is differentially overexpressed in human cancer as compared with normal tissues, in vivo. Acute silencing of Ran in various tumor cell types causes aberrant mitotic spindle formation, mitochondrial dysfunction, and apoptosis. This pathway does not require p53, Bax, or Smac, but is controlled by survivin as a novel Ran target in cancer. Conversely, loss of Ran in normal cells is well tolerated and does not result in mitotic defects or loss of cell viability. Therefore, tumor cells can become dependent on Ran signaling for cell division, and targeting this pathway may provide a novel and selective anticancer strategy. [Cancer Res 2008;68(6):1826–33]
Proteinase-Activated Receptor-1–Triggered Activation of Tumor Progression Locus-2 Promotes Actin Cytoskeleton Reorganization and Cell Migration
Authors: Hatziapostolou Maria , Polytarchou Christos , Panutsopulos Dimitrios , Covic Lidija , Tsichlis Philip N.
Published: March 2008

Tumor progression locus 2 (Tpl2), a mitogen-activated protein kinase kinase kinase (MAP3K) that is activated by provirus insertion in retrovirus-induced rodent lymphomas and mammary adenocarcinomas, is known to transduce Toll-like receptor, interleukin 1, tumor necrosis factor α, and CD40 signals and to play an important role in inflammation. Here we show that Tpl2 is also required for the transduction of cell migration and gene expression signals originating in the G-protein–coupled receptor proteinase-activated receptor 1 (PAR1). PAR1 signals transduced by Tpl2 activate Rac1 and focal adhesion kinase, and they are required for reorganization of the actin cytoskeleton and cell migration. PAR1 expressed in fibroblasts can be triggered by proteinases produced by tumor cells, and PAR1 expressed in tumor cells can be triggered by proteinases produced by fibroblasts. These data suggest that signals that regulate cell migration and gene expression flow between stromal and tumor cells in both directions and that Tpl2 plays a pivotal role in this process. [Cancer Res 2008;68(6):1851–61]
Genome-Wide Analysis of the Homeobox C6 Transcriptional Network in Prostate Cancer
Authors: McCabe Colleen D. , Spyropoulos Demetri D. , Martin David , Moreno Carlos S.
Published: March 2008

Homeobox transcription factors are developmentally regulated genes that play crucial roles in tissue patterning. Homeobox C6 (HOXC6) is overexpressed in prostate cancers and correlated with cancer progression, but the downstream targets of HOXC6 are largely unknown. We have performed genome-wide localization analysis to identify promoters bound by HOXC6 in prostate cancer cells. This analysis identified 468 reproducibly bound promoters whose associated genes are involved in functions such as cell proliferation and apoptosis. We have complemented these data with expression profiling of prostates from mice with homozygous disruption of the Hoxc6 gene to identify 31 direct regulatory target genes of HOXC6. We show that HOXC6 directly regulates expression of bone morphogenic protein 7, fibroblast growth factor receptor 2, insulin-like growth factor binding protein 3, and platelet-derived growth factor receptor α (PDGFRA) in prostate cells and indirectly influences the Notch and Wnt signaling pathways in vivo. We further show that inhibition of PDGFRA reduces proliferation of prostate cancer cells, and that overexpression of HOXC6 can overcome the effects of PDGFRA inhibition. HOXC6 regulates genes with both oncogenic and tumor suppressor activities as well as several genes such as CD44 that are important for prostate branching morphogenesis and metastasis to the bone microenvironment. [Cancer Res 2008;68(6):1988–96]
Rictor and Integrin-Linked Kinase Interact and Regulate Akt Phosphorylation and Cancer Cell Survival
Authors: McDonald Paul C. , Oloumi Arusha , Mills Julia , Dobreva Iveta , Maidan Mykola , Gray Virginia , Wederell Elizabeth D. , Bally Marcel B. , Foster Leonard J. , Dedhar Shoukat
Published: March 2008

An unbiased proteomic screen to identify integrin-linked kinase (ILK) interactors revealed rictor as an ILK-binding protein. This finding was interesting because rictor, originally identified as a regulator of cytoskeletal dynamics, is also a component of mammalian target of rapamycin complex 2 (mTORC2), a complex implicated in Akt phosphorylation. These functions overlap with known ILK functions. Coimmunoprecipitation analyses confirmed this interaction, and ILK and rictor colocalized in membrane ruffles and leading edges of cancer cells. Yeast two-hybrid assays showed a direct interaction between the NH2- and COOH-terminal domains of rictor and the ILK kinase domain. Depletion of ILK and rictor in breast and prostate cancer cell lines resulted in inhibition of Akt Ser473 phosphorylation and induction of apoptosis, whereas, in several cell lines, depletion of mTOR increased Akt phosphorylation. Akt and Ser473P-Akt were detected in ILK immunoprecipitates and small interfering RNA–mediated depletion of rictor, but not mTOR, inhibited the amount of Ser473P-Akt in the ILK complex. Expression of the NH2-terminal (1–398 amino acids) rictor domain also resulted in the inhibition of ILK-associated Akt Ser473 phosphorylation. These data show that rictor regulates the ability of ILK to promote Akt phosphorylation and cancer cell survival. [Cancer Res 2008;68(6):1618–24]