bluuebunny/crossref_metadata_2025 huggingface dataset:
On the Role of the SP1 Domain in HIV-1 Particle Assembly: a Molecular Switch?
Authors: Datta Siddhartha A. K. , Temeselew Lakew G. , Crist Rachael M. , Soheilian Ferri , Kamata Anne , Mirro Jane , Harvin Demetria , Nagashima Kunio , Cachau Raul E. , Rein Alan
Published: February 2011

Expression of a retroviral protein, Gag, in mammalian cells is sufficient for assembly of immature virus-like particles (VLPs). VLP assembly is mediated largely by interactions between the capsid (CA) domains of Gag molecules but is facilitated by binding of the nucleocapsid (NC) domain to nucleic acid. We have investigated the role of SP1, a spacer between CA and NC in HIV-1 Gag, in VLP assembly. Mutational analysis showed that even subtle changes in the first 4 residues of SP1 destroy the ability of Gag to assemble correctly, frequently leading to formation of tubes or other misassembled structures rather than proper VLPs. We also studied the conformation of the CA-SP1 junction region in solution, using both molecular dynamics simulations and circular dichroism. Consonant with nuclear magnetic resonance (NMR) studies from other laboratories, we found that SP1 is nearly unstructured in aqueous solution but undergoes a concerted change to an α-helical conformation when the polarity of the environment is reduced by addition of dimethyl sulfoxide (DMSO), trifluoroethanol, or ethanol. Remarkably, such a coil-to-helix transition is also recapitulated in an aqueous medium at high peptide concentrations. The exquisite sensitivity of SP1 to mutational changes and its ability to undergo a concentration-dependent structural transition raise the possibility that SP1 could act as a molecular switch to prime HIV-1 Gag for VLP assembly. We suggest that changes in the local environment of SP1 when Gag oligomerizes on nucleic acid might trigger this switch.
Hepatitis C Patient-Derived Glycoproteins Exhibit Marked Differences in Susceptibility to Serum Neutralizing Antibodies: Genetic Subtype Defines Antigenic but Not Neutralization Serotype
Authors: Tarr Alexander W. , Urbanowicz Richard A. , Hamed Mohamed R. , Albecka Anna , McClure C. Patrick , Brown Richard J. P. , Irving William L. , Dubuisson Jean , Ball Jonathan K.
Published: February 2011

Neutralizing antibodies have a role in controlling hepatitis C virus (HCV) infection. A successful vaccine will need to elicit potently neutralizing antibodies that are capable of preventing the infection of genetically diverse viral isolates. However, the specificity of the neutralizing antibody response in natural HCV infection still is poorly understood. To address this, we examined the reactivity of polyclonal antibodies isolated from chronic HCV infection to the diverse patient-isolated HCV envelope glycoproteins E1 and E2 (E1E2), and we also examined the potential to neutralize the entry of pseudoparticles bearing these diverse E1E2 proteins. The genetic type of the infection was found to determine the pattern of the antibody recognition of these E1E2 proteins, with the greatest reactivity to homologous E1E2 proteins. This relationship was strongest when the component of the antibody response directed only to linear epitopes was analyzed. In contrast, the neutralization serotype did not correlate with genotype. Instead, serum-derived antibodies displayed a range of neutralization breadth and potency, while different E1E2 glycoproteins displayed different sensitivities to neutralization, such that these could be divided broadly into neutralization-sensitive and -resistant phenotypes. An important additional observation was that entry mediated by some E1E2 proteins was enhanced in the presence of some of the polyclonal antibody fractions isolated during chronic infection. These data highlight the need to use diverse E1E2 isolates, which represent extremes of neutralization sensitivity, when screening antibodies for therapeutic potential and for testing antibodies generated following immunization as part of vaccine development.
The Epstein-Barr Virus BRRF1 Protein, Na, Induces Lytic Infection in a TRAF2- and p53-Dependent Manner
Authors: Hagemeier Stacy R. , Barlow Elizabeth A. , Kleman Ariel A. , Kenney Shannon C.
Published: February 2011

The Epstein-Barr virus (EBV) BRRF1 lytic gene product (Na) is encoded within the same immediate-early region as the BZLF1 (Z) and BRLF1(R) gene products, but its role during EBV infection has not been well defined. We previously showed that Na cooperates with the R protein to induce lytic gene expression in latently infected EBV-positive 293 cells, and in some EBV-negative cell lines it can activate the Z promoter in reporter gene assays. Here we show that overexpression of Na alone is sufficient to induce lytic gene expression in several different latently infected epithelial cell lines (Hone-Akata, CNE2-Akata, and AGS-Akata), while knockdown of endogenous Na expression reduces lytic gene expression. Consistent with its ability to interact with tumor necrosis factor receptor-associated factor 2 (TRAF2) in a yeast two-hybrid assay, we demonstrate that Na interacts with TRAF2 in cells. Furthermore, we show that TRAF2 is required for Na induction of lytic gene expression, that Na induces Jun N-terminal protein kinase (JNK) activation in a TRAF2-dependent manner, and that a JNK inhibitor abolishes the ability of Na to disrupt viral latency. Additionally, we show that Na and the tumor suppressor protein p53 cooperate to induce lytic gene expression in epithelial cells (including the C666-1 nasopharyngeal carcinoma cell line), although Na does not appear to affect p53 function. Together these data suggest that Na plays an important role in regulating the switch between latent and lytic infection in epithelial cells and that this effect requires both the TRAF2 and p53 cellular proteins.
Unmasking the Active Helicase Conformation of Nonstructural Protein 3 from Hepatitis C Virus
Authors: Ding Steve C. , Kohlway Andrew S. , Pyle Anna M.
Published: February 2011

The nonstructural protein 3 (NS3) helicase/protease is an important component of the hepatitis C virus (HCV) replication complex. We hypothesized that a specific β-strand tethers the C terminus of the helicase domain to the protease domain, thereby maintaining HCV NS3 in a compact conformation that differs from the extended conformations observed for other
Flaviviridae
NS3 enzymes. To test this hypothesis, we removed the β-strand and explored the structural and functional attributes of the truncated NS3 protein (NS3ΔC7). Limited proteolysis, hydrodynamic, and kinetic measurements indicate that NS3ΔC7 adopts an extended conformation that contrasts with the compact form of the wild-type (WT) protein. The extended conformation of NS3ΔC7 allows the protein to quickly form functional complexes with RNA unwinding substrates. We also show that the unwinding activity of NS3ΔC7 is independent of the substrate 3′-overhang length, implying that a monomeric form of the protein promotes efficient unwinding. Our findings indicate that an open, extended conformation of NS3 is required for helicase activity and represents the biologically relevant conformation of the protein during viral replication.
Genome Structure of Caulobacter Phage phiCb5
Authors: Kazaks Andris , Voronkova Tatyana , Rumnieks Janis , Dishlers Andris , Tars Kaspars
Published: February 2011

The complete genome sequence of caulobacter phage phiCb5 has been determined, and four open reading frames (ORFs) have been identified and characterized. As for related phages, the ORFs code for maturation, coat, replicase, and lysis proteins, but unlike other
Leviviridae
members, the lysis protein gene of phiCb5 entirely overlaps with the replicase in a different reading frame. The lysis protein of phiCb5 is about two times longer than that of the distantly related MS2 phage and presumably contains two transmembrane helices. Analysis of the proposed genome secondary structure revealed a stable 5′ stem-loop, similar to other phages, and a substantially shorter 3′ untranslated region (UTR) structure with only three stem-loops.
Distinct Severe Acute Respiratory Syndrome Coronavirus-Induced Acute Lung Injury Pathways in Two Different Nonhuman Primate Species
Authors: Smits Saskia L. , van den Brand Judith M. A. , de Lang Anna , Leijten Lonneke M. E. , van IJcken Wilfred F. , van Amerongen Geert , Osterhaus Albert D. M. E. , Andeweg Arno C. , Haagmans Bart L.
Published: February 2011

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), caused by influenza A virus H5N1 and severe acute respiratory syndrome coronavirus (SARS-CoV), supposedly depend on activation of the oxidative-stress machinery that is coupled with innate immunity, resulting in a strong proinflammatory host response. Inflammatory cytokines, such as interleukin 1β (IL-1β), IL-8, and IL-6, play a major role in mediating and amplifying ALI/ARDS by stimulating chemotaxis and activation of neutrophils. To obtain further insight into the pathogenesis of SARS-CoV-associated ALI, we compared SARS-CoV infections in two different nonhuman primate species, cynomolgus macaques and African green monkeys. Viral titers in the upper and lower respiratory tract were not significantly different in SARS-CoV-infected macaques and African green monkeys. Inflammatory cytokines that play a major role in mediating and amplifying ALI/ARDS or have neutrophil chemoattractant activity, such as IL-6, IL-8, CXCL1, and CXCL2, were, however, induced only in macaques. In contrast, other proinflammatory cytokines and chemokines, including osteopontin and CCL3, were upregulated in the lungs of African green monkeys to a significantly greater extent than in macaques. Because African green monkeys developed more severe ALI than macaques, with hyaline membrane formation, some of these differentially expressed proinflammatory genes may be critically involved in development of the observed pathological changes. Induction of distinct proinflammatory genes after SARS-CoV infection in different nonhuman primate species needs to be taken into account when analyzing outcomes of intervention strategies in these species.
Consistent Inhibition of HIV-1 Replication in CD4
+
T Cells by Acyclovir without Detection of Human Herpesviruses
Authors: McMahon Moira A. , Parsons Teresa L. , Shen Lin , Siliciano Janet D. , Siliciano Robert F.
Published: February 2011

Acyclovir, a nucleoside analog, is thought to be specific for the human herpesviruses because it requires a virally encoded enzyme to phosphorylate it to acyclovir monophosphate. Recently, acyclovir triphosphate was shown to be a direct inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase. Here, we showed that acyclovir is an inhibitor of HIV-1 replication in CD4
+
T cells from cord blood that have undetectable levels of the eight human herpesviruses. Additionally, acyclovir phosphates were detected by reverse-phase-high performance liquid chromatography (RP-HPLC) and quantified in a primer extension assay from cord blood. The data support acyclovir as an inhibitor of HIV-1 replication in herpesvirus-negative cells.
The Unique Transmembrane Hairpin of Flavivirus Fusion Protein E Is Essential for Membrane Fusion
Authors: Fritz Richard , Blazevic Janja , Taucher Christian , Pangerl Karen , Heinz Franz X. , Stiasny Karin
Published: February 2011

The fusion of enveloped viruses with cellular membranes is mediated by proteins that are anchored in the lipid bilayer of the virus and capable of triggered conformational changes necessary for driving fusion. The flavivirus envelope protein E is the only known viral fusion protein with a double membrane anchor, consisting of two antiparallel transmembrane helices (TM1 and TM2). TM1 functions as a stop-transfer sequence and TM2 as an internal signal sequence for the first nonstructural protein during polyprotein processing. The possible role of this peculiar C-terminal helical hairpin in membrane fusion has not been investigated so far. We addressed this question by studying TM mutants of tick-borne encephalitis virus (TBEV) recombinant subviral particles (RSPs), an established model system for flavivirus membrane fusion. The engineered mutations included the deletion of TM2, the replacement of both TM domains (TMDs) by those of the related Japanese encephalitis virus (JEV), and the use of chimeric TBEV-JEV membrane anchors. Using these mutant RSPs, we provide evidence that TM2 is not just a remnant of polyprotein processing but, together with TM1, plays an active role in fusion. None of the TM mutations, including the deletion of TM2, affected early steps of the fusion process, but TM interactions apparently contribute to the stability of the postfusion E trimer and the completion of the merger of the membranes. Our data provide evidence for both intratrimer and intertrimer interactions mediated by the TMDs of E and thus extend the existing models of flavivirus membrane fusion.
Vesicular Stomatitis Virus-Based H5N1 Avian Influenza Vaccines Induce Potent Cross-Clade Neutralizing Antibodies in Rhesus Macaques
Authors: Schwartz Jennifer A. , Buonocore Linda , Suguitan Amorsolo , Hunter Meredith , Marx Preston A. , Subbarao Kanta , Rose John K.
Published: February 2011

We analyzed the ability of a vaccine vector based on vesicular stomatitis virus (VSV) to induce a neutralizing antibody (NAb) response to avian influenza viruses (AIVs) in rhesus macaques. Animals vaccinated with vectors expressing either strain A/Hong Kong/156/1997 or strain A/Vietnam/1203/2004 H5 hemagglutinin (HA) were able to generate robust NAb responses. The ability of the vectors to induce NAbs against homologous and heterologous AIVs after a single dose was dependent upon the HA antigen incorporated into the VSV vaccine. The vectors expressing strain A/Vietnam/1203/2004 H5 HA were superior to those expressing strain A/Hong Kong/156/1997 HA at inducing cross-clade NAbs.
Specific Regulation of the Chemokine Response to Viral Hemorrhagic Septicemia Virus at the Entry Site
Authors: Montero Jana , Garcia Jessica , Ordas M. Camino , Casanova Isabel , Gonzalez Antonia , Villena Alberto , Coll Julio , Tafalla Carolina
Published: February 2011

The fin bases constitute the main portal of rhabdovirus entry into rainbow trout (
Oncorhynchus mykiss
), and replication in this first site strongly conditions the outcome of the infection. In this context, we studied the chemokine response elicited in this area in response to viral hemorrhagic septicemia virus (VHSV), a rhabdovirus. Among all the rainbow trout chemokine genes studied, only the transcription levels of CK10 and CK12 were significantly upregulated in response to VHSV. As the virus had previously been shown to elicit a much stronger chemokine response in internal organs, we compared the effect of VHSV on the gills, another mucosal site which does not constitute the main site of viral entry or rhabdoviral replication. In this case, a significantly stronger chemokine response was triggered, with CK1, CK3, CK9, and CK11 being upregulated in response to VHSV and CK10 and CK12 being down-modulated by the virus. We then conducted further experiments to understand how these different chemokine responses of mucosal tissues could correlate with their capacity to support VHSV replication. No viral replication was detected in the gills, while at the fin bases, only the skin and the muscle were actively supporting viral replication. Within the skin, viral replication took place in the dermis, while viral replication was blocked within epidermal cells at some point before protein translation. The different susceptibilities of the different skin layers to VHSV correlated with the effect that VHSV has on their capacity to secrete chemotactic factors. Altogether, these results suggest a VHSV interference mechanism on the early chemokine response at its active replication sites within mucosal tissues, a possible key process that may facilitate viral entry.
KCa1.1 channel contributes to cell excitability in unmyelinated but not myelinated rat vagal afferents
Authors: Li Bai-Yan , Glazebrook Patricia , Kunze Diana L. , Schild John H.
Published: February 2011

High conductance calcium-activated potassium (BKCa) channels can modulate cell excitability and neurotransmitter release at synaptic and afferent terminals. BKCa channels are present in primary afferents of most, if not, all internal organs and are an intriguing target for pharmacological manipulation of visceral sensation. Our laboratory has a long-standing interest in the neurophysiological differences between myelinated and unmyelinated visceral afferent function. Here, we seek to determine whether there is a differential distribution of BKCa channels in myelinated and unmyelinated vagal afferents. Immunocytochemistry studies with double staining for the BK-type KCa1.1 channel protein and isolectin B4 (IB4), a reliable marker of unmyelinated peripheral afferents, reveal a pattern of IB4 labeling that strongly correlates with the expression of the KCa1.1 channel protein. Measures of cell size and immunostaining intensity for KCa1.1 and IB4 cluster into two statistically distinct ( P < 0.05) populations of cells. Smaller diameter neurons most often presented with strong IB4 labeling and are presumed to be unmyelinated ( n = 1,390) vagal afferents. Larger diameter neurons most often lacked or exhibited a very weak IB4 labeling and are presumed to be myelinated ( n = 58) vagal afferents. Complimentary electrophysiological studies reveal that the BKCa channel blockers charybdotoxin (ChTX) and iberiotoxin (IbTX) bring about a comparable elevation in excitability and action potential widening in unmyelinated neurons but had no effect on the excitability of myelinated vagal afferents. This study is the first to demonstrate using combined immunohistochemical and electrophysiological techniques that KCa1.1 channels are uniquely expressed in unmyelinated C-type vagal afferents and do not contribute to the dynamic discharge characteristics of myelinated A-type vagal afferents. This unique functional distribution of BK-type KCa channels may provide an opportunity for afferent selective pharmacological intervention across a wide range of visceral pathophysiologies, particularly those with a reflexogenic etiology and pain.
Age-related impairment of T cell-induced skeletal muscle precursor cell function
Authors: Dumke Breanna R. , Lees Simon J.
Published: February 2011

Sarcopenia is the age-associated loss of skeletal muscle mass and strength. Recent evidence suggests that an age-associated loss of muscle precursor cell (MPC) functionality contributes to sarcopenia. The objectives of the present study were to examine the influence of activated T cells on MPCs and determine whether an age-related defect in this signaling occurs. MPCs were collected from the gastrocnemius and plantaris of 3-mo-old (young) and 32-mo-old (old) animals. Splenic T cells were harvested using anti-CD3 Dynabead isolation. T cells were activated for 48 h with costimulation of 100 IU/ml interleukin-2 (IL-2) and 5 μg/ml of anti-CD28. Costimulation increased 5-bromo-2′-deoxyuridine incorporation of T cells from 13.4 ± 4.6% in control to 64.8 ± 6.0% in costimulated cells. Additionally, T cell cytokines increased proliferation on MPCs isolated from young muscle by 24.0 ± 5.7%, whereas there was no effect on MPCs isolated from aged muscle. T cell cytokines were also found to be a chemoattractant. T cells were able to promote migration of MPCs isolated from young muscle; however, MPCs isolated from aged muscle did not respond to the T cell-released chemokines. Conversely, whereas T cell-released cytokines did not affect myogenesis of MPCs isolated from young animals, there was a decrease in MPCs isolated from old animals. These data suggest that T cells may play a critical role in mediating MPC function. Furthermore, aging may alter T cell-induced MPC function. These findings have implications for developing strategies aimed at increasing MPC migration and proliferation leading to an improved regenerative capacity of aged skeletal muscle.
PDGF-induced vascular smooth muscle cell proliferation is associated with dysregulation of insulin receptor substrates
Authors: Zhao Yan , Biswas Swarajit K. , McNulty Patrick H. , Kozak Mark , Jun John Y. , Segar Lakshman
Published: February 2011

In vascular smooth muscle cells (VSMCs), platelet-derived growth factor (PDGF) plays a major role in inducing phenotypic switching from contractile to proliferative state. Importantly, VSMC phenotypic switching is also determined by the phosphorylation state/expression levels of insulin receptor substrate (IRS), an intermediary signaling component that is shared by insulin and IGF-I. To date, the roles of PDGF-induced key proliferative signaling components including Akt, p70S6kinase, and ERK1/2 on the serine phosphorylation/expression of IRS-1 and IRS-2 isoforms remain unclear in VSMCs. We hypothesize that PDGF-induced VSMC proliferation is associated with dysregulation of insulin receptor substrates. Using human aortic VSMCs, we demonstrate that prolonged PDGF treatment led to sustained increases in the phosphorylation of protein kinases such as Akt, p70S6kinase, and ERK1/2, which mediate VSMC proliferation. In addition, PDGF enhanced IRS-1/IRS-2 serine phosphorylation and downregulated IRS-2 expression in a time- and concentration-dependent manner. Notably, phosphoinositide 3-kinase (PI 3-kinase) inhibitor (PI-103) and mammalian target of rapamycin inhibitor (rapamycin), which abolished PDGF-induced Akt and p70S6kinase phosphorylation, respectively, blocked PDGF-induced IRS-1 serine phosphorylation and IRS-2 downregulation. In contrast, MEK1/ERK inhibitor (U0126) failed to block PDGF-induced IRS-1 serine phosphorylation and IRS-2 downregulation. PDGF-induced IRS-2 downregulation was prevented by lactacystin, an inhibitor of proteasomal degradation. Functionally, PDGF-mediated IRS-1/IRS-2 dysregulation resulted in the attenuation of insulin-induced IRS-1/IRS-2-associated PI 3-kinase activity. Pharmacological inhibition of PDGF receptor tyrosine kinase with imatinib prevented IRS-1/IRS-2 dysregulation and restored insulin receptor signaling. In conclusion, strategies to inhibit PDGF receptors would not only inhibit neointimal growth but may provide new therapeutic options to prevent dysregulated insulin receptor signaling in VSMCs in nondiabetic and diabetic states.
A single prior bout of exercise protects against palmitate-induced insulin resistance despite an increase in total ceramide content
Authors: Thrush A. Brianne , Harasim Ewa , Chabowski Adrian , Gulli Roberto , Stefanyk Leslie , Dyck David J.
Published: February 2011

Ceramide accumulation has been implicated in the impairment of insulin-stimulated glucose transport in skeletal muscle following saturated fatty acid (FA) exposure. Importantly, a single bout of exercise can protect against acute lipid-induced insulin resistance. The mechanism by which exercise protects against lipid-induced insulin resistance is not completely known but may occur through a redirection of FA toward triacylglycerol (TAG) and away from ceramide and diacylglycerol (DAG). Therefore, in the current study, an in vitro preparation was used to examine whether a prior bout of exercise could confer protection against palmitate-induced insulin resistance and whether the pharmacological [50 μM fumonisin B1(FB1)] inhibition of ceramide synthesis in the presence of palmitate could mimic the protective effect of exercise. Soleus muscle of sedentary (SED), exercised (EX), and SED in the presence of FB1 (SED+FB1) were incubated with or without 2 mM palmitate for 4 h. This 2-mM palmitate exposure impaired insulin-stimulated glucose transport (−28%, P < 0.01) and significantly increased ceramide, DAG, and TAG accumulation in the SED group ( P < 0.05). A single prior bout of exercise prevented the detrimental effects of palmitate on insulin signaling and caused a partial redistribution of FA toward TAG ( P < 0.05). However, the net increase in ceramide content in response to palmitate exposure in the EX group was not different compared with SED, despite the maintenance of insulin sensitivity. The incubation of soleus from SED rats with FB1 (SED+FB1) prevented the detrimental effects of palmitate and caused a redirection of FA toward TAG accumulation ( P < 0.05). Therefore, this research suggests that although inhibiting ceramide accumulation can prevent the detrimental effects of palmitate, a single prior bout of exercise appears to protect against palmitate-induced insulin resistance, which may be independent of changes in ceramide content.
Maturation of sleep homeostasis in developing rats: a role for preoptic area neurons
Authors: Gvilia Irma , Suntsova Natalia , Angara Bryan , McGinty Dennis , Szymusiak Ronald
Published: February 2011

The present study evaluated the hypothesis that developmental changes in hypothalamic sleep-regulatory neuronal circuits contribute to the maturation of sleep homeostasis in rats during the fourth postnatal week. In a longitudinal study, we quantified electrographic measures of sleep during baseline and in response to sleep deprivation (SD) on postnatal days 21/29 (P21/29) and P22/30 ( experiment 1). During 24-h baseline recordings on P21, total sleep time (TST) during the light and dark phases did not differ significantly. On P29, TST during the light phase was significantly higher than during the dark phase. Mean duration of non-rapid-eye-movement (NREM) sleep bouts was significantly longer on P29 vs. P21, indicating improved sleep consolidation. On both P22 and P30, rats exhibited increased NREM sleep amounts and NREM electroencephalogram delta power during recovery sleep (RS) compared with baseline. Increased NREM sleep bout length during RS was observed only on P30. In experiment 2, we quantified activity of GABAergic neurons in median preoptic nucleus (MnPN) and ventrolateral preoptic area (VLPO) during SD and RS in separate groups of P22 and P30 rats using c-Fos and glutamic acid decarboxylase (GAD) immunohistochemistry. In P22 rats, numbers of Fos+GAD+ neurons in VLPO did not differ among experimental conditions. In P30 rats, Fos+GAD+ counts in VLPO were elevated during RS. MnPN neuronal activity was state-dependent in P22 rats, but Fos+GAD+ cell counts were higher in P30 rats. These findings support the hypothesis that functional emergence of preoptic sleep-regulatory neurons contributes to the maturation of sleep homeostasis in the developing rat brain.
Effect of protein source in diets fed during gestation and lactation on food intake regulation in male offspring of Wistar rats
Authors: Jahan-mihan Alireza , Smith Chris E. , Anderson G. Harvey
Published: February 2011

We hypothesized that protein source in the nutritionally adequate AIN-93G diets fed during gestation, lactation, and weaning influences food intake (FI) regulation in male offspring of Wistar rats. Pregnant rats were fed the recommended casein-based (C) or soy protein-based (S) diet during gestation ( experiment 1) or during gestation and lactation ( experiment 2). Pups (n = 12 per group) weaned to C or S diets were followed for 9 wk ( experiment 1) or 14 wk ( experiment 2). At termination, body weight was 5.4% and 9.4% higher, respectively, in offspring of dams fed the S diet. Altered FI regulation was shown by failure of devazepide (a CCK-A receptor blocker) to block FI reduction after protein preloads in offspring of S diet-fed dams, whereas it had a strong effect on offspring of C diet-fed dams ( P < 0.005). Similarly, naloxone (an opioid receptor blocker) blocked FI reduction more after casein than after soy protein preloads ( P < 0.01). In experiment 2, offspring of dams fed the S diet had higher hypothalamic gene expression of agouti related protein at weaning ( P < 0.05), and higher FI was found throughout postweaning ( P < 0.0001). FI reduction after protein preloads at week 7 and after glucose preloads at week 13 was greater in offspring of C diet-fed dams ( P < 0.05). Plasma insulin at weaning and insulin, ghrelin, and glucagon-like peptide-1 at week 15 were higher in offspring of S diet-fed dams (all P < 0.05). In conclusion, nutritionally complete C and S diets consumed during gestation and lactation differ in their effects on body weight and FI regulation in the offspring. Extending the diet from gestation alone to throughout gestation and lactation exaggerated the adverse effects of the S diet. However, the diet consumed postweaning had little effect on the outcome.
Neuronostatin acts in brain to biphasically increase mean arterial pressure through sympatho-activation followed by vasopressin secretion: the role of melanocortin receptors
Authors: Yosten Gina L. C. , Pate Alicia T. , Samson Willis K.
Published: February 2011

Neuronostatin is a recently described neuropeptide that is derived from the somatostatin preprohormone. We have shown previously that neuronostatin led to a biphasic, dose-related increase in mean arterial pressure when injected into the lateral cerebroventricle of adult, male rats. Because neuronostatin depolarized both magnocellular and parvocellular, paraventricular nucleus neurons in hypothalamic slice preparations, we hypothesized that neuronostatin elevated mean arterial pressure first by stimulating sympathetic nervous system activity followed by the release of a pressor hormone, specifically vasopressin. We found that the first phase of neuronostatin-induced increase in mean arterial pressure was reversed by pretreatment with phentolamine, indicating that phase 1 was, indeed, due to an increase in sympathetic activity. We also found that centrally injected neuronostatin led to a dose-related increase in vasopressin secretion in a time course consistent with the peak of the second phase. Furthermore, the second phase of arterial pressure elevation was reversed by pretreatment with a vasopressin 1 receptor antagonist, indicating that phase 2 was likely due to an increase in vasopressin secretion. We previously have shown that the anorexigenic and antidipsogenic effects of neuronostatin were reversed by pretreatment with the melanocortin 3/4 receptor antagonist, SHU9119, so we evaluated the ability of SHU9119 to reverse the effects of neuronostatin on MAP and vasopressin secretion. We found that SHU9119 abrogated the second phase of neuronostatin-induced increase in MAP and neuronostatin-induced vasopressin secretion, indicating that neuronostatin acts through the central melanocortin system to increase vasopressin release, ultimately leading to an elevation in MAP.
High-end arteriolar resistance limits uterine artery blood flow and restricts fetal growth in preeclampsia and gestational hypertension at high altitude
Authors: Browne Vaughn A. , Toledo-Jaldin Lilian , Davila R. Daniela , Lopez Luis P. , Yamashiro Henry , Cioffi-Ragan Darleen , Julian Colleen G. , Wilson Megan J. , Bigham Abigail W. , Shriver Mark D. , Honigman Benjamin , Vargas Enrique , Roach Robert , Moore Lorna G.
Published: February 2011

The reduction in infant birth weight and increased frequency of preeclampsia (PE) in high-altitude residents have been attributed to greater placental hypoxia, smaller uterine artery (UA) diameter, and lower UA blood flow (QUA). This cross-sectional case-control study determined UA, common iliac (CI), and external iliac (EI) arterial blood flow in Andeans residing at 3,600–4,100 m, who were either nonpregnant (NP, n = 23), or experiencing normotensive pregnancies (NORM; n = 155), preeclampsia (PE, n = 20), or gestational hypertension (GH, n = 12). Pregnancy enlarged UA diameter to ∼0.62 cm in all groups, but indices of end-arteriolar vascular resistance were higher in PE or GH than in NORM. QUAwas lower in early-onset (≤34 wk) PE or GH than in NORM, but was normal in late-onset (>34 wk) illness. Left QUAwas consistently greater than right in NORM, but the pattern reversed in PE. Although QCIand QEIwere higher in PE and GH than NORM, the fraction of QCIdistributed to the UA was reduced 2- to 3-fold. Women with early-onset PE delivered preterm, and 43% had stillborn small for gestational age (SGA) babies. Those with GH and late-onset PE delivered at term but had higher frequencies of SGA babies (GH=50%, PE=46% vs. NORM=15%, both P < 0.01). Birth weight was strongly associated with reduced QUA( R2= 0.80, P < 0.01), as were disease severity and adverse fetal outcomes. We concluded that high end-arteriolar resistance, not smaller UA diameter, limited QUAand restricted fetal growth in PE and GH. These are, to our knowledge, the first quantitative measurements of QUAand pelvic blood flow in early- vs. late-onset PE in high-altitude residents.
Increased renal dopamine and acute renal adaptation to a high-phosphate diet
Authors: Weinman Edward J. , Biswas Rajatsubhra , Steplock Deborah , Wang Peili , Lau Yuen-Sum , Desir Gary V. , Shenolikar Shirish
Published: February 2011

The current experiments explore the role of dopamine in facilitating the acute increase in renal phosphate excretion in response to a high-phosphate diet. Compared with a low-phosphate (0.1%) diet for 24 h, mice fed a high-phosphate (1.2%) diet had significantly higher rates of phosphate excretion in the urine associated with a two- to threefold increase in the dopamine content of the kidney and in the urinary excretion of dopamine. Animals fed a high-phosphate diet had a significant increase in the abundance and activity of renal DOPA (l-dihydroxyphenylalanine) decarboxylase and significant reductions in renalase, monoamine oxidase A, and monoamine oxidase B. The activity of protein kinase A and protein kinase C, markers of activation of renal dopamine receptors, were significantly higher in animals fed a high-phosphate vs. a low-phosphate diet. Treatment of rats with carbidopa, an inhibitor of DOPA decarboxylase, impaired adaptation to a high-phosphate diet. These experiments indicate that the rapid adaptation to a high-phosphate diet involves alterations in key enzymes involved in dopamine synthesis and degradation, resulting in increased renal dopamine content and activation of the signaling cascade used by dopamine to inhibit the renal tubular reabsorption of phosphate.
Binaural processing by the gecko auditory periphery
Authors: Christensen-Dalsgaard Jakob , Tang Yezhong , Carr Catherine E.
Published: February 2011

Lizards have highly directional ears, owing to strong acoustical coupling of the eardrums and almost perfect sound transmission from the contralateral ear. To investigate the neural processing of this remarkable tympanic directionality, we combined biophysical measurements of eardrum motion in the Tokay gecko with neurophysiological recordings from the auditory nerve. Laser vibrometry shows that their ear is a two-input system with approximately unity interaural transmission gain at the peak frequency (∼1.6 kHz). Median interaural delays are 260 μs, almost three times larger than predicted from gecko head size, suggesting interaural transmission may be boosted by resonances in the large, open mouth cavity ( Vossen et al. 2010 ). Auditory nerve recordings are sensitive to both interaural time differences (ITD) and interaural level differences (ILD), reflecting the acoustical interactions of direct and indirect sound components at the eardrum. Best ITD and click delays match interaural transmission delays, with a range of 200–500 μs. Inserting a mold in the mouth cavity blocks ITD and ILD sensitivity. Thus the neural response accurately reflects tympanic directionality, and most neurons in the auditory pathway should be directional.
Synaptic transmission and plasticity are modulated by nonmuscle myosin II at the neuromuscular junction ofDrosophila
Authors: Seabrooke Sara , Stewart Bryan A.
Published: February 2011

The synaptic vesicle population in a nerve terminal is traditionally divided into subpopulations according to physiological criteria; the readily releasable pool (RRP), the recycling pool, and the reserve pool. It is recognized that the RRP subserves synaptic transmission evoked by low-frequency neural activity and that the recycling and reserve populations are called on to supply vesicles as neural activity increases. Here we investigated the contribution of nonmuscle myosin II (NMMII) to synaptic transmission with emphasis on the role a motor protein could play in the supply of vesicles. We used Drosophila genetics to manipulate NMMII and assessed synaptic transmission at the larval neuromuscular junction. We observed a positive correlation between synaptic strength at low-frequency stimulation and NMMII expression: reducing NMMII reduced the evoked response, while increasing NMMII increased the evoked response. Further, we found that NMMII contributed to the spontaneous release of vesicles differentially from evoked release, suggesting differential contribution to these two release mechanisms. By measuring synaptic responses under conditions of differing external calcium concentration in saline, we found that NMMII is important for normal synaptic transmission under high-frequency stimulation. This research identifies diverse functions for NMMII in synaptic transmission and suggests that this motor protein is an active contributor to the physiology of synaptic vesicle recruitment.
Gastric and pyloric motor pattern control by a modulatory projection neuron in the intact crabCancer pagurus
Authors: Hedrich Ulrike B. S. , Diehl Florian , Stein Wolfgang
Published: February 2011

Neuronal release of modulatory substances provides motor pattern generating circuits with a high degree of flexibility. In vitro studies have characterized the actions of modulatory projection neurons in great detail in the stomatogastric nervous system, a model system for neuromodulatory influences on central pattern generators. Less is known about the activities and actions of modulatory neurons in fully functional and richly modulated network settings, i.e., in intact animals. It is also unknown whether their activities contribute to the motor patterns in different behavioral conditions. Here, we show for the first time the activity and effects of the well-characterized modulatory projection neuron 1 (MCN1) in vivo and compare them to in vitro conditions. MCN1 was always spontaneously active, typically in a rhythmic fashion with its firing being interrupted by ascending inhibitions from the pyloric motor circuit. Its activity contributed to pyloric motor activity, because 1) the cycle period of the motor pattern correlated with MCN1 firing frequency and 2) stimulating MCN1 shortened the cycle period while 3) lesioning of the MCN1 axon reduced motor activity. In addition, gastric mill motor activity was elicited for the duration of the stimulation. Chemosensory stimulation of the antennae moved MCN1 away from baseline activity by increasing its firing frequency. Following this increase, a gastric mill rhythm was elicited and the pyloric cycle period decreased. Lesioning the MCN1 axon prevented these effects. Thus modulatory projection neurons such as MCN1 can control the motor output in vivo, and they participate in the processing of exteroceptive sensory information in behaviorally relevant conditions.
Preclinical Evaluation of Telomerase-Specific Oncolytic Virotherapy for Human Bone and Soft Tissue Sarcomas
Authors: Sasaki Tsuyoshi , Tazawa Hiroshi , Hasei Jo , Kunisada Toshiyuki , Yoshida Aki , Hashimoto Yuuri , Yano Shuya , Yoshida Ryosuke , Uno Futoshi , Kagawa Shunsuke , Morimoto Yuki , Urata Yasuo , Ozaki Toshifumi , Fujiwara Toshiyoshi
Published: February 2011

Purpose: Tumor-specific replication-selective oncolytic virotherapy is a promising antitumor therapy for induction of cell death in tumor cells but not of normal cells. We previously developed an oncolytic adenovirus, OBP-301, that kills human epithelial malignant cells in a telomerase-dependent manner. Recent evidence suggests that nonepithelial malignant cells, which have low telomerase activity, maintain telomere length through alternative lengthening of telomeres (ALT). However, it remains unclear whether OBP-301 is cytopathic for nonepithelial malignant cells. Here, we evaluated the antitumor effect of OBP-301 on human bone and soft tissue sarcoma cells.

Experimental Design: The cytopathic activity of OBP-301, coxsackie and adenovirus receptor (CAR) expression, and telomerase activity were examined in 10 bone (OST, U2OS, HOS, HuO9, MNNG/HOS, SaOS-2, NOS-2, NOS-10, NDCS-1, and OUMS-27) and in 4 soft tissue (CCS, NMS-2, SYO-1, and NMFH-1) sarcoma cell lines. OBP-301 antitumor effects were assessed using orthotopic tumor xenograft models. The fiber-modified OBP-301 (termed OBP-405) was used to confirm an antitumor effect on OBP-301–resistant sarcomas.

Results: OBP-301 was cytopathic for 12 sarcoma cell lines but not for the non–CAR-expressing OUMS-27 and NMFH-1 cells. Sensitivity to OBP-301 was dependent on CAR expression and not on telomerase activity. ALT-type sarcomas were also sensitive to OBP-301 because of upregulation of human telomerase reverse transcriptase (hTERT) mRNA following virus infection. Intratumoral injection of OBP-301 significantly suppressed the growth of OST and SYO-1 tumors. Furthermore, fiber-modified OBP-405 showed antitumor effects on OBP-301–resistant OUMS-27 and NMFH-1 cells.

Conclusions: A telomerase-specific oncolytic adenovirus is a promising antitumor reagent for the treatment of bone and soft tissue sarcomas. Clin Cancer Res; 17(7); 1828–38. ©2011 AACR.
Rapid Diagnosis of Medulloblastoma Molecular Subgroups
Authors: Schwalbe Ed C. , Lindsey Janet C. , Straughton Debbie , Hogg Twala L. , Cole Michael , Megahed Hisham , Ryan Sarra L. , Lusher Meryl E. , Taylor Michael D. , Gilbertson Richard J. , Ellison David W. , Bailey Simon , Clifford Steven C.
Published: February 2011

Purpose: Microarray studies indicate medulloblastoma comprises distinct molecular disease subgroups, which offer potential for improved clinical management.

Experimental Design: Minimal mRNA expression signatures diagnostic for the Wnt/Wingless (WNT) and Sonic Hedgehog (SHH) subgroups were developed, validated, and used to assign subgroup affiliation in 173 tumors from four independent cohorts, alongside a systematic investigation of subgroup clinical and molecular characteristics.

Results: WNT tumors [12% (21/173)] were diagnosed >5 years of age (peak, 10 years), displayed classic histology, CTNNB1 mutation (19/20), and associated chromosome 6 loss, and have previously been associated with favorable prognosis. SHH cases [24% (42/173)] predominated in infants (<3 years) and showed an age-dependent relationship to desmoplastic/nodular pathology; all infant desmoplastic/nodular cases (previously associated with a good outcome) were SHH-positive, but these relationships broke down in noninfants. PTCH1 mutations were common [34% (11/32)], but PTCH1 exon1c hypermethylation, chromosome 9q and REN (KCTD11) genetic loss were not SHH associated, and SMO or SUFU mutation, PTCH1 exon1a or SUFU hypermethylation did not play a role, indicating novel activating mechanisms in the majority of SHH cases. SHH tumors were associated with an absence of COL1A2 methylation. WNT/SHH-independent medulloblastomas [64% (110/173)] showed all histologies, peaked at 3 and 6 years, and were exclusively associated with chromosome 17p loss.

Conclusions: Medulloblastoma subgroups are characterized by distinct genomic, epigenomic and clinicopathologic features, and clinical outcomes. Validated array-independent gene expression assays for the rapid assessment of subgroup affiliation in small biopsies provide a basis for their routine clinical application, in strategies including molecular disease-risk stratification and delivery of targeted therapeutics. Clin Cancer Res; 17(7); 1883–94. ©2011 AACR.
Reduced p21WAF1/CIP1 via Alteration of p53-DDX3 Pathway Is Associated with Poor Relapse-Free Survival in Early-Stage Human Papillomavirus–Associated Lung Cancer
Authors: Wu De-Wei , Liu Wen-Shan , Wang John , Chen Chih-Yi , Cheng Ya-Wen , Lee Huei
Published: February 2011

Purpose: DDX3 alteration has been shown to participate in hepatocellular tumorigenesis via p21WAF1/CIP1 (p21) deregulation. We observed that DDX3 and p21 expression in lung tumors was negatively associated with E6 expression. Therefore, the aim of this study was to clarify whether deregulation of p21 by DDX3 via an E6-inactivated p53 pathway would enhance tumor progression in HPV-associated lung cancers.

Experimental Design: Real-time PCR, luciferase assays, immunoprecipitation, and chromatin immunoprecipitation (ChIP) were performed to determine whether DDX3 was regulated by p53 to synergistically enhance p21 transcriptional activity. Cell proliferation was examined by cell counting and colony formation assays. DDX3 and p21 expression were evaluated in 138 lung tumors by real-time PCR and immunohistochemistry. The prognostic value of p21 expression on relapse-free survival (RFS) was analyzed by Kaplan–Meier analysis.

Results: Real-time PCR, luciferase assays, and ChIP assays indicated that three putative p53 binding sites, located at −1,080/−1,070, −695/−685, and −283/−273 on the DDX3 promoter, were required for DDX3 transcription. DDX3 deregulation by the E6-inactivated p53 pathway could promote cell proliferation and the ability to form colonies via reduced Sp1 binding activity on the p21 promoter. Among tumors, p21 expression was positively associated with DDX3 expression and negatively related with E6 expression, particularly in early-stage (I + II) tumors. Interestingly, low p21 expression was associated with a poor RFS in early-stage lung cancer.

Conclusion: The reduction of p21 by the alteration of the p53-DDX3 pathway plays an essential role in early-stage HPV-associated lung tumorigenesis and is correlated with poor RFS of lung cancer patients. Clin Cancer Res; 17(7); 1895–905. ©2011 AACR.
Reduction of Ovarian and Oviductal Cancers in Calorie-Restricted Laying Chickens
Authors: Carver Donna K. , Barnes H. John , Anderson Kenneth E. , Petitte James N. , Whitaker Regina , Berchuck Andrew , Rodriguez Gustavo C.
Published: February 2011

Epithelial ovarian cancer (OVAC) remains a highly lethal malignancy. It is a leading cause of cancer deaths among women in the United States causing more deaths than all other gynecologic malignancies combined. The pathogenesis of OVAC is not completely understood, but the process of repeated ovulation is believed to lead to genetic damage in the ovarian epithelium. As part of a prospective trial designed to evaluate OVAC chemopreventive strategies using the chicken model, caloric restriction (55% less energy) was used to inhibit ovulation in groups of hens receiving chemopreventives, thereby minimizing the impact of ovulation on the incidence of reproductive tract cancer. A separate group of chickens was maintained concurrently in the same environment, and managed similarly, except that caloric intake was not restricted. Among birds not receiving chemopreventive agents, we compared caloric versus noncaloric restricted birds to determine the relations between calorie restriction and risk of developing adenocarcinoma of the reproductive tract. Mortality in the calorie-restricted group was almost half that of those on full feed. Calorie-restricted chickens maintained body weights averaging 1.423 kg compared with the full-fed birds at 1.892 kg. Ovulation rate varied with the full-fed group producing 64% more eggs than the calorie-restricted group. Total reproductive cancers occurred in 57 (33.3%) birds for the full-fed group and 26 (10.3%) birds for the calorie-restricted group. On the basis of histopathology, 45 (26.3%) birds in the full-fed group had ovarian adenocarcinoma compared with 16 (6.3%) birds in the calorie-restricted group. Calorie restriction in laying hens resulted in a near five-fold reduction in OVAC. Cancer Prev Res; 4(4); 562–7. ©2011 AACR.