bluuebunny/crossref_metadata_2025 huggingface dataset:
Efficient Replication of Human Immunodeficiency Virus Type 1 in Resting CD4+T Lymphocytes Is Induced by Coculture with Autologous Dendritic Cells in the Absence of Foreign Antigens
Authors: Barat Corinne , Gilbert Caroline , Tremblay Michel J.
Published: December 2008

Dendritic cells (DC) are considered to be important contributors to human immunodeficiency virus type 1 (HIV-1) transmission and pathogenesis. As the first target cells in mucosal tissues, they can be become productively infected and can also capture virions and transfer them efficiently to CD4+T cells located within lymphoid tissues. Resting CD4+T cells appear to be another major target of HIV-1 in vivo, yet several blocks restrict replication in such cells. We report here that physical contact between virus-infected quiescent CD4+T cells and uninfected autologous immature DC in the absence of any foreign antigen relieves these restrictions, allowing a highly productive HIV-1 replication.
The Chromodomain of Tf1 Integrase Promotes Binding to cDNA and Mediates Target Site Selection
Authors: Chatterjee Atreyi Ghatak , Leem Young Eun , Kelly Felice D. , Levin Henry L.
Published: December 2008

The long terminal repeat (LTR) retrotransposon Tf1 of
Schizosaccharomyces pombe
integrates specifically into the promoters of pol II-transcribed genes. Its integrase (IN) contains a C-terminal chromodomain related to the chromodomains that bind to the N-terminal tail of histone H3. Although we have been unable to detect an interaction between histone tails and the chromodomain of Tf1 IN, it is possible that the chromodomain plays a role in directing IN to its target sites. To test this idea, we generated transposons with single amino acid substitutions in highly conserved residues of the chromodomain and created a chromodomain-deleted mutant. The mutations, V1290A, Y1292A, W1305A, and CHDΔ, substantially reduced transposition activity in vivo. Blotting assays showed that there was little or no reduction in the levels of IN or cDNA. By measuring the homologous recombination between cDNA and the plasmid copy of Tf1, we found that two of the mutations did not reduce the import of cDNA into the nucleus, while another caused a 33% reduction. Chromatin immunoprecipitation assays revealed that CHDΔ caused an approximately threefold reduction in the binding of IN to the downstream LTR of the cDNA. These data indicate that the chromodomain contributed directly to integration. We therefore tested whether the chromodomain contributed to selecting insertion sites. Results of a target plasmid assay showed that the deletion of the chromodomain resulted in a drastic reduction in the preference for pol II promoters. Collectively, these data indicate that the chromodomain promotes binding of cDNA and plays a key role in efficient targeting.
Orthopoxviruses Require a Functional Ubiquitin-Proteasome System for Productive Replication
Authors: Teale Alastair , Campbell Stephanie , Van Buuren Nick , Magee Wendy C. , Watmough Kelly , Couturier Brianne , Shipclark Robyn , Barry Michele
Published: December 2008

Cellular homeostasis depends on an intricate balance of protein expression and degradation. The ubiquitin-proteasome pathway plays a crucial role in specifically targeting proteins tagged with ubiquitin for destruction. This degradation can be effectively blocked by both chemically synthesized and natural proteasome inhibitors. Poxviruses encode a number of proteins that exploit the ubiquitin-proteasome system, including virally encoded ubiquitin molecules and ubiquitin ligases, as well as BTB/kelch proteins and F-box proteins, which interact with cellular ubiquitin ligases. Here we show that poxvirus infection was dramatically affected by a range of proteasome inhibitors, including MG132, MG115, lactacystin, and bortezomib (Velcade). Confocal microscopy demonstrated that infected cells treated with MG132 or bortezomib lacked viral replication factories within the cytoplasm. This was accompanied by the absence of late gene expression and DNA replication; however, early gene expression occurred unabated. Proteasomal inhibition with MG132 or bortezomib also had dramatic effects on viral titers, severely blocking viral replication and propagation. The effects of MG132 on poxvirus infection were reversible upon washout, resulting in the production of late genes and viral replication factories. Significantly, the addition of an ubiquitin-activating enzyme (E1) inhibitor had a similar affect on late and early protein expression. Together, our data suggests that a functional ubiquitin-proteasome system is required during poxvirus infection.
Tax1BP1 Interacts with Papillomavirus E2 and Regulates E2-Dependent Transcription and Stability
Authors: Wang Xiaoyu , Naidu Samisubbu R. , Sverdrup Francis , Androphy Elliot J.
Published: December 2008

The papillomavirus E2 proteins regulate viral replication, gene transcription, and genome maintenance by interacting with other viral and host proteins. From a yeast two-hybrid screen, we identified the cellular protein Tax1BP1 as a novel binding partner of human papillomavirus type 18 (HPV18) E2. Tax1BP1 also interacts with the HPV16 and bovine papillomavirus type 1 (BPV1) E2 proteins, with the C-terminal region of Tax1BP1 interacting with the N-terminal transactivation domain of BPV1 E2. Tax1BP1 complexes with p300 and acts synergistically as a coactivator with p300 to enhance E2-dependent transcription. Using chromatin immunoprecipitation assays, we show that Tax1BP1 and E2 localize to the long control region on the BPV1 genome. Tax1BP1 was recently reported to bind ubiquitin and to function as an essential component of an A20 ubiquitin-editing complex. We demonstrate that Tax1BP1 plays a role in the regulation of the steady-state level of E2 by preventing its proteasomal degradation. These studies provide new insights into the regulation of E2 functions.
Arsenic Trioxide Inhibits Hepatitis C Virus RNA Replication through Modulation of the Glutathione Redox System and Oxidative Stress
Authors: Kuroki Misao , Ariumi Yasuo , Ikeda Masanori , Dansako Hiromichi , Wakita Takaji , Kato Nobuyuki
Published: December 2008

Arsenic trioxide (ATO), a therapeutic reagent used for the treatment of acute promyelocytic leukemia, has recently been reported to increase human immunodeficiency virus type 1 infectivity. However, in this study, we have demonstrated that replication of genome-length hepatitis C virus (HCV) RNA (O strain of genotype 1b) was notably inhibited by ATO at submicromolar concentrations without cell toxicity. RNA replication of HCV-JFH1 (genotype 2a) and the release of core protein into the culture supernatants were also inhibited by ATO after the HCV infection. To clarify the mechanism of the anti-HCV activity of ATO, we examined whether or not PML is associated with this anti-HCV activity, since PML is known to be a target of ATO. Interestingly, we observed the cytoplasmic translocation of PML after treatment with ATO. However, ATO still inhibited the HCV RNA replication even in the PML knockdown cells, suggesting that PML is dispensable for the anti-HCV activity of ATO. In contrast, we found thatN-acetyl-cysteine, an antioxidant and glutathione precursor, completely and partially eliminated the anti-HCV activity of ATO after 24 h and 72 h of treatment, respectively. In this context, it is worth noting that we found an elevation of intracellular superoxide anion radical, but not hydrogen peroxide, and the depletion of intracellular glutathione in the ATO-treated cells. Taken together, these findings suggest that ATO inhibits the HCV RNA replication through modulation of the glutathione redox system and oxidative stress.
Regulation of APOBEC3 Proteins by a Novel YXXL Motif in Human Immunodeficiency Virus Type 1 Vif and Simian Immunodeficiency Virus SIVagm Vif
Authors: Pery Erez , Rajendran Kottampatty S. , Brazier Andrew Jay , Gabuzda Dana
Published: December 2008

The APOBEC3 cytidine deaminases are potent antiviral factors that restrict the replication of human immunodeficiency virus type 1 (HIV-1). In HIV-1-infected CD4
+
T cells, the viral accessory protein Vif binds to APOBEC3G (A3G), APOBEC3F (A3F), and APOBEC3C (A3C) and targets these proteins for polyubiquitination by forming an E3 ubiquitin ligase with cullin 5. Previous studies identified regions of HIV-1 Vif,
40
YRHHY
44
and
12
QVDRMR
17
, which are important for interaction with A3G and A3F, respectively, and showed that Vif residues 54 to 71 are sufficient for A3G binding. Here, we identify
69
YXXL
72
as a novel conserved motif in HIV-1 Vif that mediates binding to human A3G and its subsequent degradation. Studies on other APOBEC3 proteins revealed that Tyr69 and Leu72 are important for the degradation of A3F and A3C as well. Similar to A3F, A3C regulation is also mediated by Vif residues
12
QVDRMR
17
. Simian immunodeficiency virus (SIV) Vif was shown to bind and degrade African green monkey A3G (agmA3G) and, unexpectedly, human A3C. The YXXL motif of SIVagm Vif was important for the inactivation of agmA3G and human A3C. Unlike HIV-1 Vif, however, SIVagm Vif does not require Tyr40 and His43 for agmA3G degradation. Tyr69 in the YXXL motif was critical for binding of recombinant glutathione
S
-transferase-Vif(1-94) to A3G in vitro. These results suggest that the YXXL motif in Vif is a potential target for small-molecule inhibitors to block Vif interaction with A3G, A3F, and A3C, and thereby protect cells against HIV-1 infection.
Host Responses to Wild-Type and Attenuated Herpes Simplex Virus Infection in the Absence of Stat1
Authors: Pasieka Tracy Jo , Cilloniz Cristian , Lu Betty , Teal Thomas H. , Proll Sean C. , Katze Michael G. , Leib David A.
Published: December 2008

Humans and mice lacking the interferon signaling molecule Stat1 are sensitive to a variety of pathogens due to their presumed inability to mount a strong innate immune response. The herpes simplex virus type 1 (HSV-1) virion host shutoff (vhs) protein is a multifunctional immunomodulator that counteracts the innate immune response and viruses lacking vhs are attenuated and effective live vaccines in animal models. To investigate the interplay of viruses with an immunocompromised host, we performed functional genomics analyses on control and Stat1−/−mouse corneas infected with wild-type or vhs-null viruses. In control mice, correlative with viral growth, both viruses induced a transient increase in immunomodulators, followed by viral clearance. In contrast, infection of the Stat1−/−mice induced a heightened and prolonged induction of inflammatory modulators for both viruses, manifesting as a significant immune cell infiltrate and ocular disease. Moreover, while wild-type virus infection of Stat1−/−was always lethal, vhs-null infection was rarely lethal. There was a significant increase in Stat3- and interleukin-6 (IL-6)-dependent transcription in Stat1−/−mice, implicating the Stat3 and IL-6 pathways in the observed ocular pathology. Further, infected Stat1−/−mice showed phosphorylated Stat3 in the corneal epithelium. Our data show a role for vhs in evading innate host responses and a role for Stat1 in limiting virus infection and for facilitating an appropriate nonpathological inflammatory response.
Role of Conserved Cysteines in the Alphavirus E3 Protein
Authors: Parrott Megan M. , Sitarski Sarah A. , Arnold Randy J. , Picton Lora K. , Hill R. Blake , Mukhopadhyay Suchetana
Published: December 2008

Alphavirus particles are covered by 80 glycoprotein spikes that are essential for viral entry. Spikes consist of the E2 receptor binding protein and the E1 fusion protein. Spike assembly occurs in the endoplasmic reticulum, where E1 associates with pE2, a precursor containing E3 and E2 proteins. E3 is a small, cysteine-rich, extracellular glycoprotein that mediates proper folding of pE2 and its subsequent association with E1. In addition, cleavage of E3 from the assembled spike is required to make the virus particles efficiently fusion competent. We have found that the E3 protein in Sindbis virus contains one disulfide bond between residues Cys19 and Cys25. Replacing either of these two critical cysteines resulted in mutants with attenuated titers. Replacing both cysteines with either alanine or serine resulted in double mutants that were lethal. Insertion of additional cysteines based on E3 proteins from other alphaviruses resulted in either sequential or nested disulfide bond patterns. E3 sequences that formed sequential disulfides yielded virus with near-wild-type titers, while those that contained nested disulfide bonds had attenuated activity. Our data indicate that the role of the cysteine residues in E3 is not primarily structural. We hypothesize that E3 has an enzymatic or functional role in virus assembly, and these possibilities are further discussed.
Monoclonal Antibodies against the Fusion Peptide of Hemagglutinin Protect Mice from Lethal Influenza A Virus H5N1 Infection
Authors: Prabhu Nayana , Prabakaran Mookkan , Ho Hui-Ting , Velumani Sumathy , Qiang Jia , Goutama Michael , Kwang Jimmy
Published: December 2008

The HA2 glycopolypeptide (gp) is highly conserved in all influenza A virus strains, and it is known to play a major role in the fusion of the virus with the endosomal membrane in host cells during the course of viral infection. Vaccines and therapeutics targeting this HA2 gp could induce efficient broad-spectrum immunity against influenza A virus infections. So far, there have been no studies on the possible therapeutic effects of monoclonal antibodies (MAbs), specifically against the fusion peptide of hemagglutinin (HA), upon lethal infections with highly pathogenic avian influenza (HPAI) H5N1 virus. We have identified MAb 1C9, which binds to GLFGAIAGF, a part of the fusion peptide of the HA2 gp. We evaluated the efficacy of MAb 1C9 as a therapy for influenza A virus infections. This MAb, which inhibited cell fusion in vitro when administered passively, protected 100% of mice from challenge with five 50% mouse lethal doses of HPAI H5N1 influenza A viruses from two different clades. Furthermore, it caused earlier clearance of the virus from the lung. The influenza virus load was assessed in lung samples from mice challenged after pretreatment with MAb 1C9 (24 h prior to challenge) and from mice receiving early treatment (24 h after challenge). The study shows that MAb 1C9, which is specific to the antigenically conserved fusion peptide of HA2, can contribute to the cross-clade protection of mice infected with H5N1 virus and mediate more effective recovery from infection.
Genotyping Schemes for Polyomavirus BK, Using Gene-Specific Phylogenetic Trees and Single Nucleotide Polymorphism Analysis
Authors: Luo Chunqing , Bueno Marta , Kant Jeffrey , Martinson Jeremy , Randhawa Parmjeet
Published: December 2008

BK virus (BKV) genotyping has been historically based on nucleotides 1744 to 1812 in the VP1 gene. We reevaluated this practice by making BKV whole-genome and gene-specific phylogenetic trees as well as performing single nucleotide polymorphism (SNP) analysis of 162 sequences available in the public domain. It was found that currently known BKV subtypes and subgroups can no longer be reliably determined by sequencing certain partial gene sequences. Phylogenetic trees based on large T-antigen (LTA) allow separation of subtype I into subgroups Ia, Ib1, Ib2, and Ic, with bootstrap values of 100%, which are better than bootstraps obtained using VP1 sequences (bootstrap values of 71 to 97%). Subtype IV can be subdivided into subgroups, but LTA bootstrap values (33 to 80%) are lower than those obtained by whole-genome analysis (68 to 87%). Subtypes V and VI provisionally identified earlier on the basis of more limited sequence data are better classified as subgroups Ib2 and Ib1, respectively. LTA positions 3634, 3772, 3934, and 4339 can serve as a minimal SNP set to distinguish between the four major BKV subtypes. No subtype II-, IVa-, or IVb-defining SNPs are available in the VP1 gene. However, the overall congruence of viral strain classification based on either VP1 or LTA phylogenetic analysis indicates that these two areas of the viral genome are genetically linked. Interstrain genetic recombination between distant loci in the VP1 and LTA areas is not a common event.
Expanded Breadth of the T-Cell Response to Mosaic Human Immunodeficiency Virus Type 1 Envelope DNA Vaccination
Authors: Kong Wing-Pui , Wu Lan , Wallstrom Timothy C. , Fischer Will , Yang Zhi-Yong , Ko Sung-Youl , Letvin Norman L. , Haynes Barton F. , Hahn Beatrice H. , Korber Bette , Nabel Gary J.
Published: December 2008

An effective AIDS vaccine must control highly diverse circulating strains of human immunodeficiency virus type 1 (HIV-1). Among HIV-1 gene products, the envelope (Env) protein contains variable as well as conserved regions. In this report, an informatic approach to the design of T-cell vaccines directed to HIV-1 Env M group global sequences was tested. Synthetic Env antigens were designed to express mosaics that maximize the inclusion of common potential T-cell epitope (PTE) 9-mers and minimize the inclusion of rare epitopes likely to elicit strain-specific responses. DNA vaccines were evaluated using intracellular cytokine staining in inbred mice with a standardized panel of highly conserved 15-mer PTE peptides. One-, two-, and three-mosaic sets that increased theoretical epitope coverage were developed. The breadth and magnitude of T-cell immunity stimulated by these vaccines were compared to those for natural strain Envs; additional comparisons were performed on mutant Envs, including gp160 or gp145 with or without V regions and gp41 deletions. Among them, the two- or three-mosaic Env sets elicited the optimal CD4 and CD8 responses. These responses were most evident in CD8 T cells; the three-mosaic set elicited responses to an average of eight peptide pools, compared to two pools for a set of three natural Envs. Synthetic mosaic HIV-1 antigens can therefore induce T-cell responses with expanded breadth and may facilitate the development of effective T-cell-based HIV-1 vaccines.
Live Chimeric and Inactivated Japanese Encephalitis Virus Vaccines Differ in Their Cross-Protective Values against Murray Valley Encephalitis Virus
Authors: Lobigs Mario , Larena Maximilian , Alsharifi Mohammed , Lee Eva , Pavy Megan
Published: December 2008

The Japanese encephalitis virus (JEV) serocomplex, which also includes Murray Valley encephalitis virus (MVEV), is a group of antigenically closely related, mosquito-borne flaviviruses that are responsible for severe encephalitic disease in humans. While vaccines against the prominent members of this serocomplex are available or under development, it is unlikely that they will be produced specifically against those viruses which cause less-frequent disease, such as MVEV. Here we have evaluated the cross-protective values of an inactivated JEV vaccine (JE-VAX) and a live chimeric JEV vaccine (ChimeriVax-JE) against MVEV in two mouse models of flaviviral encephalitis. We show that (i) a three-dose vaccination schedule with JE-VAX provides cross-protective immunity, albeit only partial in the more severe challenge model; (ii) a single dose of ChimeriVax-JE gives complete protection in both challenge models; (iii) the cross-protective immunity elicited with ChimeriVax-JE is durable (≥5 months) and broad (also giving protection against West Nile virus); (iv) humoral and cellular immunities elicited with ChimeriVax-JE contribute to protection against lethal challenge with MVEV; (v) ChimeriVax-JE remains fully attenuated in immunodeficient mice lacking type I and type II interferon responses; and (vi) immunization with JE-VAX, but not ChimeriVax-JE, can prime heterologous infection enhancement in recipients of vaccination on a low-dose schedule, designed to mimic vaccine failure or waning of vaccine-induced immunity. Our results suggest that the live chimeric JEV vaccine will protect against other viruses belonging to the JEV serocomplex, consistent with the observation of cross-protection following live virus infections.
Selective Uptake of Small RNA Molecules in the Virion of Murine Gammaherpesvirus 68
Authors: Cliffe Anna R. , Nash Anthony A. , Dutia Bernadette M.
Published: December 2008

Noncoding RNAs are a feature of many herpesvirus genomes. They include microRNAs, whose function is the subject of intense investigation, in addition to longer RNA molecules such as the Epstein-Barr virus-encoded RNAs and herpesvirus saimiri U RNAs, which have been known for some time but whose function is still not well defined. Murine gammaherpesvirus 68 (MHV-68) encodes eight viral tRNA-like molecules (vtRNAs) of unknown function. Investigating the kinetics of expression of the vtRNAs, we observed that they were present directly after infection with the virus. This strongly suggested that vtRNAs were part of the virion structure, which was confirmed by their detection within various purified, RNase-treated preparations. Although both viral and cellular mRNAs were also detected within the MHV-68 virion, the major RNA species present were small RNAs of around 70 nucleotides in length. Interestingly, incorporation of viral mRNA was not related to the relative abundance in infected cells, as M11 mRNA, which is present at low abundance, was found in virions. MHV-76, which lacks the genes encoding the vtRNAs, also incorporated small RNA molecules within the virion, suggesting a requirement for these molecules for virion maturation. In productively infected cells the vtRNAs localized predominantly within the cytoplasm, although they also exhibited a globular pattern of nuclear staining. Their presence in the cytoplasm is consistent with interaction with virion components prior to maturation of virus particles. The significance of these findings for virion architecture and function is discussed.
Latent Species C Adenoviruses in Human Tonsil Tissues
Authors: Garnett C. T. , Talekar G. , Mahr J. A. , Huang W. , Zhang Y. , Ornelles D. A. , Gooding L. R.
Published: December 2008

Although species C human adenoviruses establish persistent infections, the molecular details of this lifestyle remain poorly understood. We previously reported that adenovirus DNA is found in human mucosal T lymphocytes in a noninfectious form (C. T. Garnett, D. Erdman, W. Xu, and L. R. Gooding, J. Virol. 76:10608-10616, 2002). In this study, human tonsil and adenoid tissues were analyzed to determine the dynamics of infection, the rate of clearance of viral DNA, and the possibility of reactivation of virus from these tissues. The presence of viral DNA peaked at 4 years of age and declined thereafter. The average number of viral genomes declined with the age of the donor. The frequency of virus-bearing cells ranged from 3 × 10−7to 3.4 × 10−4, while the amount of viral DNA per cell varied less, with an average of 280 copies per cell. All species C serotypes were represented in these tissues, although adenovirus type 6 was notably rare. Infectious virus was detected infrequently (13 of 94 of donors tested), even among donors with the highest levels of adenoviral DNA. Adenovirus transcripts were rarely detected in uncultured lymphocytes (2 of 12 donors) but appeared following stimulation and culture (11 of 13 donors). Viral DNA replication could be stimulated in most donor samples by lymphocyte stimulation in culture. New infectious virus was detected in 13 of 15 donors following in vitro stimulation. These data suggest that species C adenoviruses can establish latent infections in mucosal lymphocytes and that stimulation of these cells can cause viral reactivation resulting in RNA transcription, DNA replication, and infectious virus production.
Autonomic nervous system regulates secretion of anti-inflammatory prohormone SMR1 from rat salivary glands
Authors: Morris K. E. , St. Laurent C. D. , Hoeve R. S. , Forsythe P. , Suresh M. R. , Mathison R. D. , Befus A. D.
Published: December 2008

The autonomic nervous system regulates the secretion of bioactive proteins and peptides from salivary glands that can be important in systemic physiological responses. The prohormone submandibular rat-1, which is highly expressed in rat submandibular glands, can be cleaved to produce polypeptides with analgesic and anti-inflammatory activities. Human genes related to submandibular rat-1 have conserved biological functions and are potentially important in pain suppression, erectile function, and inflammation. In this study we describe the differential expression and posttranslational modification of submandibular rat-1 protein in salivary glands, the urogenital tract, lung, blood, and saliva in male Sprague-Dawley and Brown Norway rats. Submandibular rat-1 protein is secreted into saliva after the administration of β-adrenergic or cholinergic agonists. Removal of the sympathetic ganglion that innervates the salivary glands results in increased levels of submandibular rat-1 protein in salivary glands. The secretion of submandibular rat-1 in response to physiological stress may provide a large pool of submandibular rat-1-derived peptide products that can promote analgesia and decrease inflammation locally and systemically. This pathway may be conserved among mammals and may constitute an important anti-inflammatory and analgesic response to stress.
Simvastatin enhances bone marrow stromal cell differentiation into endothelial cells via notch signaling pathway
Authors: Xu Jian , Liu Xinfeng , Chen Jieli , Zacharek Alex , Cui Xu , Savant-Bhonsale Smita , Liu Zhenguo , Chopp Michael
Published: December 2008

Bone marrow stromal cells (BMSCs) are capable of differentiating into multiple cell lineages including endothelial cells. Simvastatin, an HMG-CoA reductase inhibitor that is used as a cholesterol-lowering agent, promotes endothelial differentiation from epithelial progenitor cells (EPC). The Notch signaling pathway, which plays a key role in multiple cell functions such as differentiation, proliferation, and apoptosis, can be regulated by simvastatin. Therefore, we examined the effect of simvastatin on BMSC differentiation into endothelial cells and the underlying mechanisms involved in this process. We observed that simvastatin stimulation of rat BMSCs resulted in significantly increased expression of endothelial-specific genes and proteins, including von Willebrand factor (vWF), CD31, vascular endothelial-cadherin (VE-cadherin), vascular endothelial growth factor receptor-2 (VEGFR2, Flk-1), and VEGF receptor 1 (VEGFR-1, Flt-1). Simvastatin also significantly increased capillary tubelike formation of the BMSCs. In addition, the intracellular cleavage of Notch (NICD) was markedly enhanced by simvastatin in BMSCs. Incubation of BMSCs with a γ-secretase inhibitor, or Notch1 small interfering RNA (siRNA) that significantly inhibited the formation of NICD, blocked the expression of endothelial-specific markers in BMSCs and their differentiation into functional endothelial cells. These data suggest that simvastatin induces rat BMSCs differentiation into endothelial cells via a Notch signaling pathway.
Overexpression of Galgt2 in skeletal muscle prevents injury resulting from eccentric contractions in both mdx and wild-type mice
Authors: Martin Paul T. , Xu Rui , Rodino-Klapac Louise R. , Oglesbay Elaine , Camboni Marybeth , Montgomery Chrystal L. , Shontz Kim , Chicoine Louis G. , Clark K. Reed , Sahenk Zarife , Mendell Jerry R. , Janssen Paul M. L.
Published: December 2008

The cytotoxic T cell (CT) GalNAc transferase, or Galgt2, is a UDP-GalNAc:β1,4- N-acetylgalactosaminyltransferase that is localized to the neuromuscular synapse in adult skeletal muscle, where it creates the synaptic CT carbohydrate antigen {GalNAcβ1,4[NeuAc(orGc)α2, 3]Galβ1,4GlcNAcβ-}. Overexpression of Galgt2 in the skeletal muscles of transgenic mice inhibits the development of muscular dystrophy in mdx mice, a model for Duchenne muscular dystrophy. Here, we provide physiological evidence as to how Galgt2 may inhibit the development of muscle pathology in mdx animals. Both Galgt2 transgenic wild-type and mdx skeletal muscles showed a marked improvement in normalized isometric force during repetitive eccentric contractions relative to nontransgenic littermates, even using a paradigm where nontransgenic muscles had force reductions of 95% or more. Muscles from Galgt2 transgenic mice, however, showed a significant decrement in normalized specific force and in hindlimb and forelimb grip strength at some ages. Overexpression of Galgt2 in muscles of young adult mdx mice, where Galgt2 has no effect on muscle size, also caused a significant decrease in force drop during eccentric contractions and increased normalized specific force. A comparison of Galgt2 and microdystrophin overexpression using a therapeutically relevant intravascular gene delivery protocol showed Galgt2 was as effective as microdystrophin at preventing loss of force during eccentric contractions. These experiments provide a mechanism to explain why Galgt2 overexpression inhibits muscular dystrophy in mdx muscles. That overexpression also prevents loss of force in nondystrophic muscles suggests that Galgt2 is a therapeutic target with broad potential applications.
Molecular beacons can assess changes in expression and 3′-polyadenylation of human eNOS mRNA
Authors: Jones Rachel , Baker Meredith B. , Weber Martina , Harrison David G. , Bao Gang , Searles Charles D.
Published: December 2008

The endothelium plays an essential role in maintaining vascular homeostasis, and it fulfills this role by modulating intracellular signaling and gene expression in response to chemical and mechanical stimuli. Assessing changes in endothelial gene expression is essential to understanding how physiological and pathophysiological processes modulate vascular homeostasis. Here we describe the use of molecular beacons to rapidly and quantitatively assess expression and 3′-polyadenylation of a gene that is important for vascular homeostasis, endothelial nitric oxide synthase (eNOS). Single- and dual-fluorescence resonance energy transfer (FRET) molecular beacon hybridization assays were developed to measure changes in mRNA levels and 3′-polyadenylation, respectively, in primary human endothelial cell cultures subjected to laminar shear stress or statin treatment. Optimized beacon hybridization assays took ∼15 min to perform, and eNOS mRNA levels were validated by quantitative real-time RT-PCR. Competitive inhibition assays and posttranscriptional silencing of eNOS expression were used to verify the specificity of molecular beacon fluorescence. Finally, the dual-FRET method was used to assess eNOS polyadenylation in tissues isolated from mice subjected to exercise training. These data demonstrate that molecular beacons can be used to rapidly and efficiently measure endothelial gene expression and 3′-polyadenylation. This approach could easily be adapted for studies of other endothelial genes and has promise for applications in live endothelial cells.
Orlistat accelerates gastric emptying and attenuates GIP release in healthy subjects
Authors: Enç Feruze Yılmaz , Öneş Tunç , Akın H. Levent , Dede Fuat , Turoğlu H. Turgut , Ülfer Gözde , Bekiroğlu Nural , Haklar Goncagül , Rehfeld Jens F. , Holst Jens J. , Ulusoy Nefise B , İmeryüz Neşe
Published: December 2008

Orlistat, an inhibitor of digestive lipases, is widely used for the treatment of obesity. Previous reports on the effect of orally ingested orlistat together with a meal on gastric emptying and secretion of gut peptides that modulate postprandial responses are controversial. We investigated the effect of ingested orlistat on gastric emptying and plasma responses of gut peptides in response to a solid mixed meal with a moderate energy load. In healthy subjects, gastric emptying was determined using scintigraphy and studies were performed without and with 120 mg of orlistat in pellet form in random order. Orlistat shortened t lag and t half and decreased the area under the gastric emptying curve. Orlistat significantly attenuated the secretion of glucose-dependent insulinotropic polypeptide (GIP) but did not alter the plasma responses of cholecystokinin (CCK), glucagon-like peptide-1 (GLP-1), pancreatic polypeptide (PP), and insulin. There was no peptide YY (PYY) response. Area under the curve of gastric emptying was positively correlated with integrated secretion of GIP ( r = 0.786) in orlistat and was negatively correlated with integrated plasma response of GLP-1 ( r = −0.75) in control experiments, implying that inhibition of fat absorption modifies determinants of gastric emptying of a meal. Orlistat administered similar to its use in obesity treatment accelerates gastric emptying of a solid mixed meal with a moderate energy load and profoundly attenuates release of GIP without appreciably altering plasma responses of CCK, GLP-1, and PP. Since GIP is being implemented in the development of obesity, its role in weight control attained by orlistat awaits further investigation.
Human intestinal intraepithelial lymphocytes and epithelial cells coinduce interleukin-8 production through the CD2-CD58 interaction
Authors: Ebert Ellen C. , Panja Asit , Praveen Rajalakshmi
Published: December 2008

Human intestinal CD3+TCRαβ+CD8+intraepithelial lymphocytes (IELs) are intimately associated with epithelial cells (ECs) through binding of CD103 to E-cadherin. How these two cell types functionally interact is largely unknown. IEL-EC cross talk was determined using HT-29 cells as the model EC and IL-8 as the readout. IL-8 was derived from both cell types and synergistically increased when the cells were combined. This synergistic effect required active transcription by both IELs and HT-29 cells. Cell contact was required as shown by the loss of the synergistic increase in IL-8 when the two cell types were separated by Transwells. Specifically, IL-8 release required the binding of CD2 on the IELs to CD58 on the HT-29 cells. The association of the CD3/TCR complex with major histocompatibility antigen class I antigens was not involved. Antibody neutralization of tumor necrosis factor-α (TNF-α), but not interferon-γ (IFN-γ), resulted in increased IL-8 production by the coculture. Although both TNF-α and IFN-γ increased IL-8 synthesis and CD58 expression by the HT-29 cells, only IFN-γ reduced IL-8 production by IELs. IL-8 production by either cell type involved phosphorylation of p38 and JNK. In summary, the synergistic synthesis of IL-8 occurs when IELs are stimulated through the CD2 pathway by CD58 on HT-29 cells, resulting in TNF-α release that, in turn, augments IL-8 synthesis and CD58 expression by the HT-29 cells.
Glucagon-like peptide-2 relaxes mouse stomach through vasoactive intestinal peptide release
Authors: Amato Antonella , Baldassano Sara , Serio Rosa , Mulè Flavia
Published: December 2008

Glucagon-like peptide-2 (GLP-2) influences different aspects of the gastrointestinal function, including epithelial growth, digestion, absorption, motility, and blood flow. Intraluminal pressure from isolated mouse stomach was recorded to investigate whether GLP-2 affects gastric tone and to analyze its mechanism of action. Regional differences between diverse parts of the stomach were also examined using circular muscular strips from fundus and antrum. In the whole stomach, GLP-2 (0.3–100 nM) produced concentration-dependent relaxation with a maximum that was about 75% of relaxation to 1 μM isoproterenol (IC50 = 2.5 nM). This effect was virtually abolished by desensitization of GLP-2 receptors or by α-chymotrypsin. The relaxant response to GLP-2 was not affected by tetrodotoxin, a blocker of neuronal voltage-dependent Na+ channels, but it was significantly reduced by ω-conotoxin GVIA, a blocker of neuronal N-type voltage-operated Ca2+ channels. Nω-nitro-l-arginine methyl ester, a blocker of nitric oxide synthase, or apamin, a blocker of Ca2+-dependent potassium channels, failed to affect the gastric response to the peptide. However, the relaxation was significantly antagonized by [Lys1,Pro2,5,Arg3,4,Tyr6]VIP7–28, a vasoactive intestinal peptide (VIP) receptor antagonist (GLP-2 maximum effect = 45% of relaxation to 1 μM isoproterenol), and virtually abolished by desensitization of the VIP receptors. GLP-2 induced concentration-dependent relaxation in carbachol-precontracted fundic strips but not in antral strips. These results provide the first experimental evidence that GLP-2 is able to induce gastric relaxation acting peripherally on the mouse stomach. The effect appears to be mediated by prejunctional neural release of VIP and confined to fundic region.
Endotoxin has acute and chronic effects on the cerebral circulation of fetal sheep
Authors: Feng Susan Y. S. , Phillips David J. , Stockx Elaine M. , Yu Victor Y. H. , Walker Adrian M.
Published: December 2008

We studied the impact of endotoxemia on cerebral blood flow (CBF), cerebral vascular resistance (CVR), and cerebral oxygen transport (O2 transport) in fetal sheep. We hypothesized that endotoxemia impairs CBF regulation and O2 transport, exposing the brain to hypoxic-ischemic injury. Responses to lipopolysaccharide (LPS; 1 μg/kg iv on 3 consecutive days, n = 9) or normal saline ( n = 5) were studied. Of LPS-treated fetuses, five survived and four died; in surviving fetuses, transient cerebral vasoconstriction at 0.5 h (ΔCVR approximately +50%) was followed by vasodilatation maximal at 5–6 h (ΔCVR approximately −50%) when CBF had increased (approximately +60%) despite reduced ABP (approximately −20%). Decreased CVR and increased CBF persisted 24 h post-LPS and the two subsequent LPS infusions. Cerebral O2 transport was sustained, although arterial O2 saturation was reduced ( P < 0.05). Histological evidence of neuronal injury was found in all surviving LPS-treated fetuses; one experienced grade IV intracranial hemorrhage. Bradykinin-induced cerebral vasodilatation (ΔCVR approximately −20%, P < 0.05) was abolished after LPS. Fetuses that died post-LPS ( n = 4) differed from survivors in three respects: CVR did not fall, CBF did not rise, and O2 transport fell progressively. In conclusion, endotoxin disrupts the cerebral circulation in two phases: 1) acute vasoconstriction (1 h) and 2) prolonged vasodilatation despite impaired endothelial dilatation (24 h). In surviving fetuses, LPS causes brain injury despite cerebral O2 transport being maintained by elevated cerebral perfusion; thus sustained O2 transport does not prevent brain injury in endotoxemia. In contrast, cerebral hypoperfusion and reduced O2 transport occur in fetuses destined to die, emphasizing the importance of sustaining O2 transport for survival.
Cerebral adaptations to chronic anemia in a model of erythropoietin-deficient mice exposed to hypoxia
Authors: El Hasnaoui-Saadani Raja , Pichon Aurélien , Marchant Dominique , Olivier Paul , Launay Thierry , Quidu Patricia , Beaudry Michèle , Duvallet Alain , Richalet Jean-Paul , Favret Fabrice
Published: December 2008

Anemia and hypoxia in rats result in an increase in factors potentially involved in cerebral angiogenesis. Therefore, the aim of this study was to assess the effect of chronic anemia and/or chronic hypoxia on cerebral cellular responses and angiogenesis in wild-type and anemic transgenic mice. These studies were done in erythropoietin-deficient mice (Epo-TAgh) in normoxia and following acute (one day) and chronic (14 days, barometric pressure = 420 mmHg) hypoxia. In normoxia, Epo-TAgh mice showed an increase in transcript and protein levels of hypoxia-inducible factor 1α (HIF-1α), vascular endothelial growth factor (VEGF), erythropoietin receptors (EpoR), phospho-STAT-5/STAT-5 ratio, and neuronal neuronal nitric oxide synthase (nNOS) along with a higher cerebral capillary density. In wild-type (WT) mice, acute hypoxia increased all of the studied factors, while in chronic hypoxia, HIF-1α, EpoR, phospho-STAT-5/STAT-5 ratio, nNOS, and inducible NOS remained elevated, with an increase in capillary density. Surprisingly, in Epo-TAgh mice, chronic hypoxia did not further increase any factor except the nitric oxide metabolites, while HIF-1α, EpoR, and phospho-STAT-5/STAT-5 ratio were reduced. Normoxic Epo-TAgh mice developed cerebral angiogenesis through the HIF-1α/VEGF pathway. In acute hypoxia, WT mice up-regulated all of the studied factors, including cerebral NO. Polycythemia and angiogenesis occurred with acclimatization to chronic hypoxia only in WT mice. In Epo-TAgh, the decrease in HIF-1α, VEGF proteins, and phospho-STAT-5 ratio in chronic hypoxia suggest that neuroprotective and angiogenesis pathways are altered.
Periodic variation in R-R intervals and cardiovascular autonomic regulation in young adult Syrian hamsters
Authors: Mongue-Din H. , Salmon A. , Fiszman M. Y. , Fromes Y.
Published: December 2008

Several hamster strains are commonly used as models for cardiomyopathic phenotypes evolving toward heart failure. However, little is known about heart rate variability (HRV) in this species. Prolonged surface ECG recording, a prerequisite to HRV studies, can be obtained either by telemetry or by restraints. Here, we performed long time ECG recording using telemetry on young adult Syrian hamsters and we analyzed time series of interbeat intervals. Standard statistics showed that the mean of normal R-R intervals slightly increased with age, with standard deviation of normal R-R intervals remaining stable over time. However, time domain analysis using Poincaré plots revealed dynamic changes in the HRV. Analysis of frequency domains revealed that the ratio of spectral components (low frequency/high frequency) exhibited a maturation pattern. Thus refined analysis of HRV revealed a more complex pattern than common statistical analysis would translate. Unlike other rodents, hamsters display a great spontaneous variability of their heart rate. As the complexity canvas of HRV might be the consequence of extracardiac regulation factors, we assessed the sympathovagal balance in both time and frequency domain of heart rate. Pharmacological tests revealed that both sympathetic and vagal tones contribute to HRV in Syrian hamsters. Thus Syrian hamsters have a broad intrinsic HRV with large influences of the neurovegetative system. However, the influence of the previous beat seems to prevail over the autonomic oscillators. These animals present a high sensitivity to artificially altered cardiac regulation and might be great models for the diagnosis of early alterations in the HRV related to pathology. Therefore, Syrian hamsters represent a unique model for HRV studies.
Mechanisms in the PVN mediating local and central sodium-induced hypertension in Wistar rats
Authors: Gabor Alexander , Leenen Frans H. H.
Published: December 2008

Sympathoexcitatory and hypertensive responses to central infusion of Na+-rich artificial cerebrospinal fluid (aCSF) are enhanced by aldosterone and mediated by mineralocorticoid receptors (MRs) and benzamil-blockable Na+influx, leading to “ouabain” release and ANG II type 1 (AT1) receptor stimulation. The present study evaluated the functional role of these mechanisms in the paraventricular nucleus (PVN). In conscious Wistar rats, Na+-rich aCSF was infused either directly into the PVN or intracerebroventricularly preceded by aldosterone and blockers. Infusion of Na+-rich aCSF in the PVN caused gradual increases in blood pressure (BP) and heart rate (HR). Aldosterone and a subpressor dose of ouabain in the PVN alone did not affect BP and HR but enhanced responses to Na+. Eplerenone, benzamil, and “ouabain”-binding Fab fragments only blocked the enhancement by aldosterone, whereas losartan blocked all responses to Na+-rich aCSF in the PVN. Increases in BP and HR by intracerebroventricular infusion of Na+-rich aCSF were enhanced by aldosterone infused intracerebroventricularly, but not in the PVN. Telmisartan in the PVN again blocked all responses. In contrast, both eplerenone and benzamil in the PVN did not change the pressor responses to intracerebroventricular infusion of aldosterone and Na+-rich aCSF. These findings indicate that AT1receptors in the PVN mediate the responses to Na+-rich aCSF and their enhancement by aldosterone, both locally in the PVN or in the general CSF. MRs, benzamil-blockable Na+channels or transporters, and “ouabain” can be functionally active in the PVN, but in Wistar rats appear not to contribute to the pressor responses to short-term increases in CSF [Na+].
Osmosensitivity of prolactin cells is enhanced by the water channel aquaporin-3 in a euryhaline Mozambique tilapia (Oreochromis mossambicus)
Authors: Watanabe Soichi , Hirano Tetsuya , Grau E. Gordon , Kaneko Toyoji
Published: December 2008

In teleost fish, prolactin (PRL) has important actions in the regulation of salt and water balances in freshwater (FW) fish. Consistent with this role, the release of PRL from the pituitary of the Mozambique tilapia is stimulated as extracellular osmolality is reduced. Stretch-activated calcium-permeant ion channels appear to be responsible for the initiation of the signal transduction that leads to increased PRL release when PRL cells are exposed to reductions in extracellular osmolality. In this study, we examined a possible involvement of the aquaporin-3 (AQP3) water channel in this osmoreceptive mechanism in PRL cells of the tilapia. AQP3 expression levels in the rostral pars distalis of the pituitary, consisting predominantly of PRL cells, were higher in fish adapted to FW than in seawater (SW)-adapted fish. Immunohistochemical studies revealed that AQP3 is located in the cell membrane and perinuclear region of PRL cells, with more intense immunosignals in PRL cells of FW-adapted fish than in those of SW fish. In FW PRL cells, the magnitude of hyposmoticity-induced cell volume increase was greater than that seen in SW PRL cells. Mercury, a potent inhibitor of AQP3, inhibited hyposmoticity-induced cell volume increase and PRL release from FW PRL cells. The inhibitory effect of mercury was partially restored by β-mercaptoethanol, whereas no effect of mercury was observed on PRL release stimulated by a depolarizing concentration of KCl, which induces Ca2+influx and stimulates the subsequent Ca2+-signaling pathway. These results indicate significant contribution of AQP3 to osmoreception in PRL cells in FW-adapted tilapia.
Reactive oxygen species-dependent endothelin signaling is required for augmented hypoxic sensory response of the neonatal carotid body by intermittent hypoxia
Authors: Pawar Anita , Nanduri Jayasri , Yuan Guoxiang , Khan Shakil A. , Wang Ning , Kumar Ganesh K. , Prabhakar Nanduri R.
Published: December 2008

We previously reported that intermittent hypoxia (IH) augments hypoxic sensory response (HSR) and increases the number of glomus cells in neonatal carotid bodies. In the present study, we tested the hypothesis that recruitment of endothelin-1 (ET-1) signaling by reactive oxygen species (ROS) plays a critical role in IH-evoked changes in neonatal carotid bodies. Experiments were performed on neonatal rats exposed either to 10 days of IH (P0–P10; 8 h/day) or to normoxia. IH augmented HSR of the carotid bodies ex vivo and resulted in hyperplasia of glomus cells. The effects of IH were associated with enhanced basal release of ET-1 under normoxia, sensitization of carotid body response to exogenous ET-1, and upregulation of ETA but not an ETB receptor mRNA without altering the ET-1 content. An ETA but not ETB receptor antagonist prevented augmented HSR by IH. ROS levels were elevated in carotid bodies from IH-treated rat pups as evidenced by increased levels of malondialdehyde. Systemic administration of manganese (III) tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride (MnTMPyP; 5 mg/kg ip), a scavenger of O2•−, prevented IH-induced elevation of ROS, basal release of ET-1, upregulation of ETA mRNA, and augmented HSR. In striking contrast, MnTMPyP treatment had no significant effect on IH-induced hyperplasia of glomus cells. These results demonstrate that IH-evoked increase in HSR involve a ROS-mediated increase in basal ET-1 release and upregulation of ETA receptor mRNA.