bluuebunny/crossref_metadata_2025 huggingface dataset:
Foundations of Societal Inequality
Authors: Acemoglu Daron , Robinson James
Published: October 2009

The degree to which economic success is passed through generations and the ability of societies to generate wealth depend on their institutions and social arrangements.
Hwang Convicted But Dodges Jail; Stem Cell Research Has Moved On
Authors: Normile Dennis
Published: October 2009

Disgraced stem cell scientist Woo Suk Hwang was handed a 2-year suspended prison sentence on 26 October for embezzlement and bioethics law violations. And the scientific community seemed to just shrug.
DOE Gives $151 Million to ‘Out-of-Box’ Research
Authors: Kintisch Eli
Published: October 2009

Three days after President Barack Obama told a Massachusetts Institute of Technology audience that he would lead the country into a "new frontier" of clean energy research, a fledgling federal agency made a $151 million down payment on that promise.
Obama's Science Advisers Look at Reform of Schools
Authors: Mervis Jeffrey
Published: October 2009

Confident that its opinions will be welcomed by the Obama Administration, the President's Council of Advisors on Science and Technology (PCAST) has launched a study of how best to improve U.S. science education.
Signs of Early
Homo sapiens
in China?
Authors: Stone Richard
Published: October 2009

A fresh find of human fossils claimed to be more than 100,000 years old challenges the prevailing view that our ancestors peopled the world in a migration out of Africa late in the last Ice Age, Chinese scientists say.
Development and Analytical Validation of an Immunoassay for Quantifying Serum Anti-Pertussis Toxin Antibodies Resulting from
Bordetella pertussis
Infection
Authors: Menzies Sandra L. , Kadwad Vijay , Pawloski Lucia C. , Lin Tsai-Lien , Baughman Andrew L. , Martin Monte , Tondella Maria Lucia C. , Meade Bruce D.
Published: October 2009

Adequately sensitive and specific methods to diagnose pertussis in adolescents and adults are not widely available. Currently, no Food and Drug Administration-approved diagnostic assays are available for the serodiagnosis of
Bordetella pertussis
. Since concentrations of
B. pertussis
-specific antibodies tend to be high during the later phases of disease, a simple, rapid, easily transferable serodiagnostic test was developed. This article describes test development, initial evaluation of a prototype kit enzyme-linked immunosorbent assay (ELISA) in an interlaboratory collaborative study, and analytical validation. The data presented here demonstrate that the kit met all prespecified criteria for precision, linearity, and accuracy for samples with anti-pertussis toxin (PT) immunoglobulin G (IgG) antibody concentrations in the range of 50 to 150 ELISA units (EU)/ml, the range believed to be most relevant for serodiagnosis. The assay met the precision and linearity criteria for a wider range, namely, from 50 to 200 EU/ml; however, the accuracy criterion was not met at 200 EU/ml. When the newly adopted World Health Organization International Standard for pertussis antiserum (human) reference reagent was used to evaluate accuracy, the accuracy criteria were met from 50 to 200 international units/ml. In conclusion, the IgG anti-PT ELISA met all assay validation parameters within the range considered most relevant for serodiagnosis. This ELISA was developed and analytically validated as a user-friendly kit that can be used in both qualitative and quantitative formats. The technology for producing the kit is transferable to public health laboratories.
Rapid Detection of
Mycobacterium tuberculosis
and Rifampin Resistance by Use of On-Demand, Near-Patient Technology
Authors: Helb Danica , Jones Martin , Story Elizabeth , Boehme Catharina , Wallace Ellen , Ho Ken , Kop JoAnn , Owens Michelle R. , Rodgers Richard , Banada Padmapriya , Safi Hassan , Blakemore Robert , Lan N. T. Ngoc , Jones-López Edward C. , Levi Michael , Burday Michele , Ayakaka Irene , Mugerwa Roy D. , McMillan Bill , Winn-Deen Emily , Christel Lee , Dailey Peter , Perkins Mark D. , Persing David H. , Alland David
Published: October 2009

Current nucleic acid amplification methods to detect
Mycobacterium tuberculosis
are complex, labor-intensive, and technically challenging. We developed and performed the first analysis of the Cepheid Gene Xpert System's MTB/RIF assay, an integrated hands-free sputum-processing and real-time PCR system with rapid on-demand, near-patient technology, to simultaneously detect
M. tuberculosis
and rifampin resistance. Analytic tests of
M. tuberculosis
DNA demonstrated a limit of detection (LOD) of 4.5 genomes per reaction. Studies using sputum spiked with known numbers of
M. tuberculosis
CFU predicted a clinical LOD of 131 CFU/ml. Killing studies showed that the assay's buffer decreased
M. tuberculosis
viability by at least 8 logs, substantially reducing biohazards. Tests of 23 different commonly occurring rifampin resistance mutations demonstrated that all 23 (100%) would be identified as rifampin resistant. An analysis of 20 nontuberculosis mycobacteria species confirmed high assay specificity. A small clinical validation study of 107 clinical sputum samples from suspected tuberculosis cases in Vietnam detected 29/29 (100%) smear-positive culture-positive cases and 33/39 (84.6%) or 38/53 (71.7%) smear-negative culture-positive cases, as determined by growth on solid medium or on both solid and liquid media, respectively.
M. tuberculosis
was not detected in 25/25 (100%) of the culture-negative samples. A study of 64 smear-positive culture-positive sputa from retreatment tuberculosis cases in Uganda detected 63/64 (98.4%) culture-positive cases and 9/9 (100%) cases of rifampin resistance. Rifampin resistance was excluded in 54/55 (98.2%) susceptible cases. Specificity rose to 100% after correcting for a conventional susceptibility test error. In conclusion, this highly sensitive and simple-to-use system can detect
M. tuberculosis
directly from sputum in less than 2 h.
Adherence Factors in Atypical Enteropathogenic
Escherichia coli
Strains Expressing the Localized Adherence-Like Pattern in HEp-2 Cells
Authors: Scaletsky Isabel C. A. , Aranda Katia R. S. , Souza Tamara B. , Silva Neusa P.
Published: October 2009

Although atypical enteropathogenic
Escherichia coli
(aEPEC) strains are frequently implicated in childhood diarrhea in developing countries, not much is known about their adherence properties. The phenotypic and genotypic characterization of 29 aEPEC strains expressing the localized adherence-like pattern points toward the involvement of
E. coli
common pilus (ECP), intimins, and other known
E. coli
adhesins in this pattern.
The Oncolytic Activity of Newcastle Disease Virus NDV-HUJ on Chemoresistant Primary Melanoma Cells Is Dependent on the Proapoptotic Activity of the Inhibitor of Apoptosis Protein Livin
Authors: Lazar Itay , Yaacov Barak , Shiloach Tamar , Eliahoo Elad , Kadouri Luna , Lotem Michal , Perlman Riki , Zakay-Rones Zichria , Panet Amos , Ben-Yehuda Dina
Published: October 2009

Patients with advanced melanoma usually do not benefit from conventional chemotherapy treatment. There is therefore a true need for a new kind of therapy for melanoma. One factor responsible for the poor prognosis of melanoma is the inhibitor of apoptosis protein (IAP) family member Livin. In this study, we applied a novel approach for the treatment of melanoma, using a unique strain of the oncolytic Newcastle disease virus (NDV-HUJ). We found that, unlike chemotherapeutic drugs, NDV-HUJ, a one-cycle replicating virus, overcomes the resistance to apoptosis of melanoma primary cultures that over express the Livin protein. In contrast, melanoma tumor cells that do not express Livin are relatively resistant to NDV-HUJ treatment. Furthermore, we show that NDV-HUJ-induced oncolysis is attributed to the dual function of Livin: although Livin inhibits apoptosis through the inhibition of caspases, under the robust apoptotic stimulation of NDV-HUJ, caspases can cleave Livin to create a truncated protein with a paradoxical proapoptotic activity. Thus, NDV-HUJ is a potent inducer of apoptosis that can overcome the antiapoptotic effect of Livin and allow cleavage of Livin into the proapoptotic tLivin protein. Moreover, the results indicate that the interferon system, which is functional in melanoma, is not involved in NDV-induced oncolysis. Taken together, our data offer the possibility of a new viral oncolytic treatment for chemoresistant melanoma.
A
trans
-Complementing Recombination Trap Demonstrates a Low Propensity of Flaviviruses for Intermolecular Recombination
Authors: Taucher Christian , Berger Angelika , Mandl Christian W.
Published: October 2009

Intermolecular recombination between the genomes of closely related RNA viruses can result in the emergence of novel strains with altered pathogenic potential and antigenicity. Although recombination between flavivirus genomes has never been demonstrated experimentally, the potential risk of generating undesirable recombinants has nevertheless been a matter of concern and controversy with respect to the development of live flavivirus vaccines. As an experimental system for investigating the ability of flavivirus genomes to recombine, we developed a “recombination trap,” which was designed to allow the products of rare recombination events to be selected and amplified. To do this, we established reciprocal packaging systems consisting of pairs of self-replicating subgenomic RNAs (replicons) derived from tick-borne encephalitis virus (TBEV), West Nile virus (WNV), and Japanese encephalitis virus (JEV) that could complement each other in
trans
and thus be propagated together in cell culture over multiple passages. Any infectious viruses with intact, full-length genomes that were generated by recombination of the two replicons would be selected and enriched by end point dilution passage, as was demonstrated in a spiking experiment in which a small amount of wild-type virus was mixed with the packaged replicons. Using the recombination trap and the JEV system, we detected two aberrant recombination events, both of which yielded unnatural genomes containing duplications. Infectious clones of both of these genomes yielded viruses with impaired growth properties. Despite the fact that the replicon pairs shared approximately 600 nucleotides of identical sequence where a precise homologous crossover event would have yielded a wild-type genome, this was not observed in any of these systems, and the TBEV and WNV systems did not yield any viable recombinant genomes at all. Our results show that intergenomic recombination can occur in the structural region of flaviviruses but that its frequency appears to be very low and that therefore it probably does not represent a major risk in the use of live, attenuated flavivirus vaccines.
Hepatitis B and Hepatitis C Virus Replication Upregulates Serine Protease Inhibitor Kazal, Resulting in Cellular Resistance to Serine Protease-Dependent Apoptosis
Authors: Lamontagne Jason , Pinkerton Mark , Block Timothy M. , Lu Xuanyong
Published: October 2009

Hepatitis B and C viruses (HBV and HCV, respectively) are different and distinct viruses, but there are striking similarities in their disease potential. Infection by either virus can cause chronic hepatitis, liver cirrhosis, and ultimately, liver cancer, despite the fact that no pathogenetic mechanisms are known which are shared by the two viruses. Our recent studies have suggested that replication of either of these viruses upregulates a cellular protein called serine protease inhibitor Kazal (SPIK). Furthermore, the data have shown that cells containing HBV and HCV are more resistant to serine protease-dependent apoptotic death. Since our previous studies have shown that SPIK is an inhibitor of serine protease-dependent apoptosis, it is hypothesized that the upregulation of SPIK caused by HBV and HCV replication leads to cell resistance to apoptosis. The evasion of apoptotic death by infected cells results in persistent viral replication and constant liver inflammation, which leads to gradual accumulation of genetic changes and eventual development of cancer. These findings suggest a possibility by which HBV and HCV, two very different viruses, can share a common mechanism in provoking liver disease and cancer.
Late Domain-Independent Rescue of a Release-Deficient Moloney Murine Leukemia Virus by the Ubiquitin Ligase Itch
Authors: Jadwin Joshua A. , Rudd Victoria , Sette Paola , Challa Swathi , Bouamr Fadila
Published: October 2009

Moloney murine leukemia virus (MoMLV) Gag utilizes its late (L) domain motif PPPY to bind members of the Nedd4-like ubiquitin ligase family. These interactions recruit components of the cell's budding machinery that are critical for virus release. MoMLV Gag contains two additional L domains, PSAP and LYPAL, that are believed to drive residual MoMLV release via interactions with cellular proteins Tsg101 and Alix, respectively. We found that overexpression of Tsg101 or Alix failed to rescue the release of PPPY-deficient MoMLV via these other L domains. However, low-level expression of the ubiquitin ligase Itch potently rescued the release and infectivity of MoMLV lacking PPPY function. In contrast, other ubiquitin ligases such as WWP1, Nedd4.1, Nedd4.2, and Nedd4.2s did not rescue this release-deficient virus. Efficient rescue required the ubiquitin ligase activity of Itch and an intact C2 domain but not presence of the endophilin-binding site. Additionally, we found Itch to immunoprecipitate with MoMLV Gag lacking the PPPY motif and to be incorporated into rescued MoMLV particles. The PSAP and LYPAL motifs were dispensable for Itch-mediated virus rescue, and their absence did not affect the incorporation of Itch into the rescued particles. Itch-mediated rescue of release-defective MoMLV was sensitive to inhibition by dominant-negative versions of ESCRT-III components and the VPS4 AAA ATPase, indicating that Itch-mediated correction of MoMLV release defects requires the integrity of the host vacuolar sorting protein pathway. RNA interference knockdown of Itch suppressed the residual release of the MoMLV lacking the PPPY motif. Interestingly, Itch stimulation of the PPPY-deficient MoMLV release was accompanied by the enhancement of Gag ubiquitination and the appearance of new ubiquitinated Gag proteins in virions. Together, these results suggest that Itch can facilitate MoMLV release in an L domain-independent manner via a mechanism that requires the host budding machinery and involves Gag ubiquitination.
Conserved Residues in the UL24 Protein of Herpes Simplex Virus 1 Are Important for Dispersal of the Nucleolar Protein Nucleolin
Authors: Bertrand Luc , Leiva-Torres Gabriel André , Hyjazie Huda , Pearson Angela
Published: October 2009

The UL24 family of proteins is widely conserved among herpesviruses. We demonstrated previously that UL24 of herpes simplex virus 1 (HSV-1) is important for the dispersal of nucleolin from nucleolar foci throughout the nuclei of infected cells. Furthermore, the N-terminal portion of UL24 localizes to nuclei and can disperse nucleolin in the absence of any other viral proteins. In this study, we tested the hypothesis that highly conserved residues in UL24 are important for the ability of the protein to modify the nuclear distribution of nucleolin. We constructed a panel of substitution mutations in UL24 and tested their effects on nucleolin staining patterns. We found that modified UL24 proteins exhibited a range of subcellular distributions. Mutations associated with a wild-type localization pattern for UL24 correlated with high levels of nucleolin dispersal. Interestingly, mutations targeting two regions, namely, within the first homology domain and overlapping or near the previously identified PD-(D/E)XK endonuclease motif, caused the most altered UL24 localization pattern and the most drastic reduction in its ability to disperse nucleolin. Viral mutants corresponding to the substitutions G121A and E99A/K101A both exhibited a syncytial plaque phenotype at 39°C. vUL24-E99A/K101A replicated to lower titers than did vUL24-G121A or KOS. Furthermore, the E99A/K101A mutation caused the greatest impairment of HSV-1-induced dispersal of nucleolin. Our results identified residues in UL24 that are critical for the ability of UL24 to alter nucleoli and further support the notion that the endonuclease motif is important for the function of UL24 during infection.
A U
L
47 Gene Deletion Mutant of Bovine Herpesvirus Type 1 Exhibits Impaired Growth in Cell Culture and Lack of Virulence in Cattle
Authors: Lobanov Vladislav A. , Maher-Sturgess Sheryl L. , Snider Marlene G. , Lawman Zoe , Babiuk Lorne A. , van Drunen Littel-van den Hurk Sylvia
Published: October 2009

Tegument protein VP8 encoded by the U
L
47 gene of bovine herpesvirus type 1 (BHV-1) is the most abundant constituent of mature virions. In the present report, we describe the characterization of U
L
47 gene-deleted BHV-1 in cultured cells and its natural host. The U
L
47 deletion mutant exhibited reduced plaque size and more than 100-fold decrease in intracellular and extracellular viral titers in cultured cells. Ultrastructural observations of infected cells showed normal maturation of BHV-1 virions in the absence of VP8. There was no evidence for a change in immediate-early gene activator function of VP16 in the U
L
47 deletion mutant virus-infected cells, since bovine ICP4 mRNA and protein levels were similar to those in the wild-type and revertant virus-infected cells throughout the course of infection. Whereas VP16, glycoprotein C (gC), gB, and VP5 were expressed to wild-type levels in the U
L
47 deletion mutant-infected cells, the gD and VP22 protein levels were significantly reduced. The reduction in gD protein was associated with increased turnover of the protein. Furthermore, some of the analyzed early and late proteins were expressed with earlier kinetics in the absence of VP8. Extracellular virions of the U
L
47 deletion mutant contained reduced amounts of gD, gB, gC, and VP22 but similar amounts of VP16 compared to those of wild-type or revertant virus particles. In addition, the U
L
47 gene product was indispensable for BHV-1 replication in vivo, since no clinical manifestations or viral shedding were detected in the U
L
47 deletion mutant-infected calves, and the virus failed to induce significant levels of humoral and cellular immunity.
Herpes Simplex Virus Proteins ICP27 and UL47 Associate with Polyadenylate-Binding Protein and Control Its Subcellular Distribution
Authors: Dobrikova Elena , Shveygert Mayya , Walters Robert , Gromeier Matthias
Published: October 2009

Human pathogenic viruses manipulate host cell translation machinery to ensure efficient expression of viral genes and to thwart host cell protein synthesis. Viral strategies include cleaving translation factors, manipulating translation factor abundance and recruitment into translation initiation complexes, or expressing viral translation factor analogs. Analyzing translation factors in herpes simplex virus type 1 (HSV-1)-infected HeLa cells, we found diminished association of the polyadenylate-binding protein (PABP) with the cap-binding complex. Although total PABP levels were unchanged, HSV-1 infection prompted accumulation of cytoplasmic PABPC1, but not its physiologic binding partner PABP-interacting protein 2 (Paip2), in the nucleus. Using glutathione
S
-transferase-PABP pull-down and proteomic analyses, we identified several viral proteins interacting with PABPC1 including tegument protein UL47 and infected-cell protein ICP27. Transient expression of ICP27 and UL47 in HeLa cells suggested that ICP27 and UL47 jointly displace Paip2 from PABP. ICP27 expression alone was sufficient to cause PABPC1 redistribution to the nucleus. ICP27 and UL47 did not alter translation efficiency of transfected reporter RNAs but modulated transcript abundance and expression of reporter cDNAs in transfected cells. This indicates that redistribution of PABPC1 may be involved in co- and posttranscriptional regulation of mRNA processing and/or nuclear export by HSV-1 gene regulatory proteins.
Vaccination To Induce Antibodies Blocking the CX3C-CX3CR1 Interaction of Respiratory Syncytial Virus G Protein Reduces Pulmonary Inflammation and Virus Replication in Mice
Authors: Zhang Wenliang , Choi Youngjoo , Haynes Lia M. , Harcourt Jennifer L. , Anderson Larry J. , Jones Les P. , Tripp Ralph A.
Published: October 2009

Respiratory syncytial virus (RSV) infection causes substantial morbidity and some deaths in the young and elderly worldwide. There is no safe and effective vaccine available, although it is possible to reduce the hospitalization rate for high-risk children by anti-RSV antibody prophylaxis. RSV has been shown to modify the immune response to infection, a feature linked in part to RSV G protein CX3C chemokine mimicry. This study determined if vaccination with G protein polypeptides or peptides spanning the central conserved region of the G protein could induce antibodies that blocked G protein CX3C-CX3CR1 interaction and disease pathogenesis mediated by RSV infection. The results show that mice vaccinated with G protein peptides or polypeptides containing the CX3C motif generate antibodies that inhibit G protein CX3C-CX3CR1 binding and chemotaxis, reduce lung virus titers, and prevent body weight loss and pulmonary inflammation. The results suggest that RSV vaccines that induce antibodies that block G protein CX3C-CX3CR1 interaction may offer a new, safe, and efficacious RSV vaccine strategy.
Insulin-Degrading Enzyme Binds to the Nonglycosylated Precursor of Varicella-Zoster Virus gE Protein Found in the Endoplasmic Reticulum
Authors: Carpenter J. E. , Jackson W. , de Souza G. A. , Haarr L. , Grose C.
Published: October 2009

Insulin degradation enzyme (IDE) is a 110-kDa zinc metalloprotease found in the cytosol of all cells. IDE degrades insulin and a variety of small proteins including amyloid-β. Recently, IDE has been proposed as the receptor for varicella-zoster virus (VZV) attachment. During our reassessment, some of the original studies were repeated and expanded in scope. We first confirmed that IDE antibody reduced VZV spread. For additional controls, we repeated the same experiments with herpes simplex virus (HSV)-infected cells as well as uninfected cells. There was a visible reduction in HSV spread but less than seen in the VZV system. Of greater importance, IDE antibody also inhibited the growth of uninfected cells. Second, we repeated the coprecipitation assays. We confirmed that antibodies to VZV gE (open reading frame 68) coprecipitated IDE and that anti-IDE antibody coprecipitated gE. However, the detected gE protein was not the mature 98-kDa form; rather, it was a precursor 73-kDa gE form found in the endoplasmic reticulum. Additional control experiments included VZV-infected cell cultures treated with tunicamycin to block gE glycosylation in the endoplasmic reticulum; again, the anti-IDE antibody coprecipitated a 73-kDa gE product. Finally, Orbitrap mass spectrometry analysis of a chromatographically purified gE sample revealed four cellular proteins associated with the unfolded protein response: BiP (HSPA5), HSPA8, HSPD1, and PPIA (peptidyl-propyl
cis
-
trans
isomerase). We conclude that IDE protease binds to the 73-kDa gE precursor and that this event occurs in the cytosol but not as a receptor/ligand interaction.
Production and Function of the Cytoplasmic Deproteinized Relaxed Circular DNA of Hepadnaviruses
Authors: Guo Haitao , Mao Richeng , Block Timothy M. , Guo Ju-Tao
Published: October 2009

Removal of genome-bound viral DNA polymerase ought to be an essential step in the formation of hepadnavirus covalently closed circular DNA (cccDNA). We previously demonstrated that deproteinized (DP) relaxed circular DNA (rcDNA) of hepatitis B virus (HBV) existed in both the cytoplasm and nuclei of infected cells and the vast majority of cytoplasmic DP rcDNA was associated with DNase I-permeable nucleocapsids. In our efforts to investigate the role of the cytoplasmic DP rcDNA in cccDNA formation, we demonstrated that rcDNA deproteinization could occur in an endogenous DNA polymerase reaction with either virion-derived or intracellular nucleocapsids. As observed in the cytoplasm of virally infected cells, in vitro deproteinization requires the maturation of plus-strand DNA and results in changes in nucleocapsid structure that render the DP rcDNA susceptible to DNase I digestion. Remarkably, we found that the cytoplasmic DP rcDNA-containing nucleocapsids could be selectively immunoprecipitated with an antibody against the carboxyl-terminal peptide of HBV core protein and are associated with cellular nuclear transport receptors karyopherin-α and -β. Moreover, transfection of small interfering RNA targeting karyopherin-β1 mRNA or expression of a dominant-negative karyopherin-β1 in a stable cell line supporting HBV replication resulted in the accumulation of DP rcDNA in cytoplasm and reduction of nuclear DP rcDNA and cccDNA. Our results thus favor a hypothesis that completion of plus-strand DNA synthesis triggers the genomic DNA deproteinization and structural changes of nucleocapsids, which leads to the exposure of nuclear localization signals in the C terminus of core protein and mediates the nuclear transportation of DP rcDNA via interaction with karyopherin-α and -β.
Generation of Live Attenuated Novel Influenza Virus A/California/7/09 (H1N1) Vaccines with High Yield in Embryonated Chicken Eggs
Authors: Chen Zhongying , Wang Weijia , Zhou Helen , Suguitan Amorsolo L. , Shambaugh Cindy , Kim Lomi , Zhao Jackie , Kemble George , Jin Hong
Published: October 2009

Several live attenuated influenza virus A/California/7/09 (H1N1) (CA09) candidate vaccine variants that possess the hemagglutinin (HA) and neuraminidase (NA) gene segments from the CA09 virus and six internal protein gene segments from the cold-adapted influenza virus A/Ann Arbor/6/60 (H2N2) virus were generated by reverse genetics. The reassortant viruses replicated relatively poorly in embryonated chicken eggs. To improve virus growth in eggs, reassortants expressing the HA and NA of CA09 were passaged in MDCK cells and variants exhibiting large-plaque morphology were isolated. These variants replicated at levels approximately 10-fold higher than the rate of replication of the parental strains in embryonated chicken eggs. Sequence analysis indicated that single amino acid changes at positions 119, 153, 154, and 186 were responsible for the improved growth properties in MDCK cells and eggs. In addition, the introduction of a mutation at residue 155 that was previously shown to enhance the replication of a 1976 swine influenza virus also significantly improved the replication of the CA09 virus in eggs. Each variant was further evaluated for receptor binding preference, antigenicity, attenuation phenotype, and immunogenicity. Mutations at residues 153, 154, and 155 drastically reduced viral antigenicity, which made these mutants unsuitable as vaccine candidates. However, changes at residues 119 and 186 did not affect virus antigenicity or immunogenicity, justifying their inclusion in live attenuated vaccine candidates to protect against the currently circulating 2009 swine origin H1N1 viruses.
META-STABLE BRANE CONFIGURATIONS WITH MULTIPLE NS5-BRANES
Authors: AHN CHANGHYUN
Published: October 2009

Starting from an [Formula: see text] supersymmetric electric gauge theory with the multiple product gauge group and the bifundamentals, we apply Seiberg dual to each gauge group, obtain the [Formula: see text] supersymmetric dual magnetic gauge theories with dual matters including the gauge singlets. Then we describe the intersecting brane configurations, where there are NS-branes and D4-branes (and anti-D4-branes), of type IIA string theory corresponding to the meta-stable nonsupersymmetric vacua of this gauge theory.

We also discuss the case where the orientifold 4-planes are added into the above brane configuration. Next, by adding an orientifold 6-plane, we apply to an [Formula: see text] supersymmetric electric gauge theory with the multiple product gauge group (where a single symplectic or orthogonal gauge group is present) and the bifundamentals. Finally, we describe the other cases where the orientifold 6-plane intersects with NS-brane.
META-STABLE BRANE CONFIGURATIONS, MULTIPLE NS5-BRANES, AND ROTATED D6-BRANES
Authors: AHN CHANGHYUN
Published: October 2009

We construct the type IIA nonsupersymmetric meta-stable brane configurations corresponding to the various [Formula: see text] supersymmetric gauge theories. The D6-branes are both displaced and rotated where these deformations are described as the mass term and quartic term for the fundamental flavors, respectively. The multiplicity of the NS5-branes occurs in the superpotential order for adjoint, symmetric, or bifundamental matters. A rich pattern of nonsupersymmetric meta-stable states as well as the supersymmetric stable states is found.
Epidermal growth factor increases lysophosphatidic acid production in human ovarian cancer cells: roles for phospholipase D2 and receptor transactivation
Authors: Snider Ashley J. , Zhang Zhihong , Xie Yuhuan , Meier Kathryn E.
Published: October 2009

Lysophosphatidic acid (LPA), is a lipid mediator that binds to G-protein coupled receptors. Epidermal growth factor (EGF), a polypeptide growth factor, binds to the EGF receptor (EGFR), a receptor tyrosine kinase. Both LPA and EGF induce responses in tumor cells that include proliferation, migration, metastasis, and induction of angiogenesis. LPA has the potential to act as an autocrine/paracrine factor and can transactivate the EGFR. This study explores the role of phospholipase D2 (PLD2) activation in LPA production, as well as cross-talk between EGF and LPA receptors. We demonstrate that EGF and LPA both stimulate production of LPA by OVCAR3 and SKOV3 human ovarian cancer cell lines. PD158780, an EGFR-selective tyrosine kinase inhibitor, blocks LPA production in response to both EGF and LPA in OVCAR3 and SKOV3 cells. Pertussis toxin, an inhibitor of LPA receptor signaling, inhibits LPA production in response to both EGF and LPA. Similar results were observed for the LPA receptor antagonist, Ki16425. Overexpression of PLD2 increases LPA production, while knockdown of PLD2 blocks EGF-induced LPA production. A phospholipase A2 (PLA2) inhibitor also blocks LPA- and EGF-induced LPA production. These results indicate that EGF stimulates LPA production in a manner that requires PLD2, and suggest that cross-talk can occur bidirectionally between EGF and LPA receptors.
Phorbol 12-myristate 13-acetate-induced endocytosis of the Na-K-2Cl cotransporter in MDCK cells is associated with a clathrin-dependent pathway
Authors: Mykoniatis Andreas , Shen Le , Fedor-Chaiken Mary , Tang Jun , Tang Xu , Worrell Roger T. , Delpire Eric , Turner Jerrold R. , Matlin Karl S. , Bouyer Patrice , Matthews Jeffrey B.
Published: October 2009

In secretory epithelial cells, the basolateral Na+-K+-2Cl− cotransporter (NKCC1) plays a major role in salt and fluid secretion. Our laboratory has identified NKCC1 surface expression as an important regulatory mechanism for Cl− secretion in the colonic crypt cell line T84, a process also present in native human colonic crypts. We previously showed that activation of protein kinase C (PKC) by carbachol and phorbol 12-myristate 13-acetate (PMA) decreases NKCC1 surface expression in T84 cells. However, the specific endocytic entry pathway has not been defined. We used a Madin-Darby canine kidney (MDCK) cell line stably transfected with enhanced green fluorescent protein (EGFP)-NKCC1 to map NKCC1 entry during PMA exposure. At given times, we fixed and stained the cells with specific markers (e.g., dynamin II, clathrin heavy chain, and caveolin-1). We also used chlorpromazine, methyl-β-cyclodextrin, amiloride, and dynasore, blockers of the clathrin, caveolin, and macropinocytosis pathways and the vesicle “pinchase” dynamin, respectively. We found that PMA caused dose- and time-dependent NKCC1 endocytosis. After 2.5 min of PMA exposure, ∼80% of EGFP-NKCC1 endocytic vesicles colocalized with clathrin and ∼40% colocalized with dynamin II and with the transferrin receptor, the uptake of which is also mediated by clathrin-coated vesicles. We did not observe significant colocalization of EGFP-NKCC1 endocytic vesicles with caveolin-1, a marker of the caveolae-mediated endocytic pathway. We quantified the effect of each inhibitor on PMA-induced EGFP-NKCC1 endocytosis and found that only chlorpromazine and dynasore caused significant inhibition compared with the untreated control (61% and 25%, respectively, at 2.5 min). Together, these results strongly support the conclusion that PMA-stimulated NKCC1 endocytosis is associated with a clathrin pathway.
Anionic leak currents through the Na+/monocarboxylate cotransporter SMCT1
Authors: Coady Michael J. , Wallendorff Bernadette , Bourgeois Francis , Lapointe Jean-Yves
Published: October 2009

SMCT1 is a Na-coupled cotransporter of short chain monocarboxylates, which is expressed in the apical membrane of diverse epithelia such as colon, renal cortex, and thyroid. We previously reported that SMCT1 cotransport was reduced by extracellular Cl− replacement with cyclamate− and that the protein exhibited an ostensible anionic leak current. In this paper, we have revisited the interaction between small monovalent anions and SMCT cotransport and leak currents. We found that the apparent Cl− dependence of cotransport was due to inhibition of this protein by the replacement anion cyclamate, whereas several other replacement anions function as substrates for SMCT1; a suitable replacement anion (MES−) was identified. The observed outward leak currents represented anionic influx and favored larger anions (NO3−>I−>Br−>Cl−); currents in excess of 1 μA (at +50 mV) could be observed and exhibited a quasilinear relationship with anion concentrations up to 100 mM. Application of 25 mM bicarbonate did not produce measurable leak currents. The leak current displayed outward rectification, which disappeared when external Na+ was replaced by N-methyl-d-glucamine+. More precisely, external Na+ blocked the leak current in both directions, but its Ki value rose rapidly when membrane potential became positive. Thus SMCT1 possesses a anionic leak current that becomes significant whenever external Na+ concentration is reduced. The presence of this leak current may represent a second function for SMCT1 in addition to cotransporting short chain fatty acids, and future experiments will determine whether this function serves a physiological role in tissues where SMCT1 is expressed.
Kupffer cell activation is a causal factor for hepatic insulin resistance
Authors: Lanthier Nicolas , Molendi-Coste Olivier , Horsmans Yves , van Rooijen Nico , Cani Patrice D. , Leclercq Isabelle A.
Published: October 2009

Recruited adipose tissue macrophages contribute to chronic and low-grade inflammation causing insulin resistance in obesity. Similarly, we hypothesized here that Kupffer cells, the hepatic resident macrophages, play a pathogenic role in hepatic insulin resistance induced by a high-fat diet. Mice were fed a normal diet or high-fat diet for 3 days. Kupffer cell activation was evaluated by immunohistochemistry and quantitative RT-PCR. Insulin sensitivity was assessed in vivo by hyperinsulinemic-euglycemic clamp and insulin-activated signaling was investigated by Western blot. Liposome-encapsulated clodronate was injected intravenously to deplete macrophages prior to a short-term exposure to high-fat diet. Here, we characterized a short-term high-fat diet model in mice and demonstrated early hepatic insulin resistance and steatosis concurrent with Kupffer cell activation. We demonstrated that selective Kupffer cell depletion obtained by intravenous clodronate, without affecting adipose tissue macrophages, was sufficient to enhance insulin-dependent insulin signaling and significantly improve hepatic insulin sensitivity in vivo in this short-term high-fat diet model. Our study clearly shows that hepatic macrophage response participates to the onset of high-fat diet-induced hepatic insulin resistance and may therefore represent an attractive target for prevention and treatment of diet- and obesity-induced insulin resistance.
Effects of environmental hypoxia on cardiac energy metabolism and performance in tilapia
Authors: Speers-Roesch Ben , Sandblom Erik , Lau Gigi Y. , Farrell Anthony P. , Richards Jeffrey G.
Published: October 2009

The ability of an animal to depress ATP turnover while maintaining metabolic energy balance is important for survival during hypoxia. In the present study, we investigated the responses of cardiac energy metabolism and performance in the hypoxia-tolerant tilapia ( Oreochromis hybrid sp.) during exposure to environmental hypoxia. Exposure to graded hypoxia (≥92% to 2.5% air saturation over 3.6 ± 0.2 h) followed by exposure to 5% air saturation for 8 h caused a depression of whole animal oxygen consumption rate that was accompanied by parallel decreases in heart rate, cardiac output, and cardiac power output (CPO, analogous to ATP demand of the heart). These cardiac parameters remained depressed by 50–60% compared with normoxic values throughout the 8-h exposure. During a 24-h exposure to 5% air saturation, cardiac ATP concentration was unchanged compared with normoxia and anaerobic glycolysis contributed to ATP supply as evidenced by considerable accumulation of lactate in the heart and plasma. Reductions in the provision of aerobic substrates were apparent from a large and rapid (in <1 h) decrease in plasma nonesterified fatty acids concentration and a modest decrease in activity of pyruvate dehydrogenase. Depression of cardiac ATP demand via bradycardia and an associated decrease in CPO appears to be an integral component of hypoxia-induced metabolic rate depression in tilapia and likely contributes to hypoxic survival.
Sex-related decrease in [Ca2+]isignaling and Ca2+-dependent contraction in inferior vena cava of female rat
Authors: Xia Yin , Khalil Raouf A.
Published: October 2009

Sex differences in the incidence of varicose veins have been suggested; however, the venous mechanisms involved are unclear. We hypothesized sex-related differences in venous function and underlying distinctions in intracellular free calcium, [Ca2+]i, signaling and Ca2+-dependent mechanisms of venous contraction. Circular segments of inferior vena cava (IVC) from male and female Sprague-Dawley rats were suspended between two hooks, labeled with fura-2, and placed in a cuvet inside a spectrofluorometer for simultaneous measurement of isometric contraction and the 340/380 fluorescence ratio (indicative of [Ca2+]i). In male IVC, phenylephrine (PHE; 10−5M) caused significant increase in contraction and [Ca2+]i. In female IVC, PHE-induced contraction was significantly reduced, but [Ca2+]idid not differ significantly from males. Membrane depolarization by KCl (96 mM), which stimulates Ca2+influx, caused parallel increases in contraction and [Ca2+]iin male IVC, and the KCl-induced contraction was significantly reduced in parallel with [Ca2+]iin female IVC. In male IVC stimulated with 0 Ca2+KCl solution, the addition of increasing concentrations of extracellular Ca2+([Ca2+]e) (0.1, 0.3, 0.6, 1, and 2.5 mM) caused stepwise increases in contraction and [Ca2+]i, and both the KCl-induced [Ca2+]e-contraction curve and the [Ca2+]e-[Ca2+]icurve were reduced in female IVC, suggesting reduced Ca2+entry via voltage-gated channels. The PHE-induced [Ca2+]e-contraction curve was significantly reduced in females, but the [Ca2+]e-[Ca2+]icurve was similar in female and male IVC, suggesting the involvement of other mechanisms in addition to Ca2+entry. The [Ca2+]e-contraction and [Ca2+]e-[Ca2+]icurves were used to construct the [Ca2+]i-contraction relationship. The KCl-induced [Ca2+]i-contraction relationship was superimposed in male and female IVC. In contrast, the PHE-induced [Ca2+]i-contraction relationship was reduced and located to the right in female compared with male IVC, suggesting reduced [Ca2+]isensitivity of the venous contractile myofilaments. The reduced contraction, [Ca2+]i, and [Ca2+]isensitivity in female veins render them more prone to dilation. These sex-specific reductions in venous function, if they also occur in human veins, may play a role in the greater incidence of varicose veins in females.
The heme oxygenase system attenuates pancreatic lesions and improves insulin sensitivity and glucose metabolism in deoxycorticosterone acetate hypertension
Authors: Ndisang Joseph Fomusi , Jadhav Ashok
Published: October 2009

Recent clinical reports indicate that impaired glucose tolerance is a common phenomenon in primary aldosteronism. Aldosterone stimulates NF-κB and activating protein-1 (AP-1) to cause oxidative injury. Elevated oxidative stress impairs insulin signaling. We recently showed that the heme oxygenase (HO) system lowers blood pressure (BP) in deoxycorticosterone-acetate (DOCA)+salt hypertension, a model of primary aldosteronism. However, the effect of the HO system on insulin sensitivity in this model remains largely unclear. Here we report the effects of the HO-inducer hemin and the HO-blocker [chromium mesoporphyrin (CrMP)] on insulin sensitivity/glucose metabolism. Our experimental design included the following 10 groups: (A) controls [(i) surgery-free or normal Sprague-Dawley (SD), (ii) uninephrectomized (UnX)-sham, (iii) UnX+salt (0.9%NaCl+0.2%KCl) and (iv) UnX+DOCA]; (B) DOCA+salt; (C) hemin+DOCA+salt; (D) hemin+CrMP+DOCA+salt; (E) CrMP+DOCA+salt; (F) vehicle-treated rats and (G) normal SD+hemin. Hemin therapy lowered BP and increased plasma insulin and the insulin-sensitizing protein adiponectin with slight but significant reduction of glycemia, while CrMP abolished the hemin effects. Furthermore, hemin improved intraperitoneal glucose and insulin tolerance, suggesting that although DOCA+salt-hypertensive rats were normoglycemic, insulin signaling may be impaired. In contrast, the HO-inhibitor CrMP aggravated insulin resistance and exacerbated glucose and insulin tolerance. Interestingly, the enhanced insulin sensitization in hemin-treated animals was accompanied by reduced urinary/gastrocnemius muscle 8-iso-prostaglandin F2α (8-isoprostane), inflammatory/oxidative transcription factors like NF-κB, AP-1, JNK, and heme content, whereas HO-1, HO-activity, cGMP, and plasma/gastrocnemius muscle antioxidants including bilirubin, ferritin, SOD, catalase, and the total antioxidant capacity were increased. Similarly, hemin enhanced pancreatic HO, cGMP, and cAMP but suppressed 8-isoprostane and attenuated pancreatic histopathological lesions including fibrosis, interstitial edema, acinar cell necrosis, vacuolization, and mononuclear cell infiltration, with corresponding improvement of insulin production. Our results suggest that impaired insulin signaling may be a forerunner to hyperglycemia in aldosteronism. By preserving pancreatic morphology, potentiating insulin signaling, and lowering BP, the HO system may prevent metabolic and cardiovascular complications in aldosteronism.
Human glomerular endothelium: interplay among glucose, free fatty acids, angiotensin II, and oxidative stress
Authors: Jaimes Edgar A. , Hua Ping , Tian Run-Xia , Raij Leopoldo
Published: October 2009

Glomerular endothelial cells (GEC) are strategically situated within the capillary loop and adjacent to the glomerular mesangium. GEC serve as targets of metabolic, biochemical, and hemodynamic signals that regulate the glomerular microcirculation. Unequivocally, hyperglycemia, hypertension, and the local renin-angiotensin system partake in the initiation and progression of diabetic nephropathy (DN). Whether free fatty acids (FFA) and reactive oxygen species (ROS) that have been associated with the endothelial dysfunction of diabetic macrovascular disease also contribute to DN is not known. Since endothelial cells from different organs and from different species may display different phenotypes, we employed human GEC to investigate the effect of high glucose (22.5 mmol/l), FFA (800 μmol/l), and angiotensin II (ANG II; 10−7 mol/l) on the genesis of ROS and their effects on endothelial nitric oxide synthase (eNOS), cyclooxygenase-2 (COX-2), and the synthesis of prostaglandins (PGs). We demonstrated that high glucose but not high FFA increased the expression of a dysfunctional eNOS as well as increased ROS from NADPH oxidase (100%) and likely from uncoupled eNOS. ANG II also induced ROS from NADPH oxidase. High glucose and ANG II upregulated (100%) COX-2 via ROS and significantly increased the synthesis of prostacyclin (PGI2) by 300%. In contrast, FFA did not upregulate COX-2 but increased PGI2 (500%). These novel studies are the first in human GEC that characterize the differential role of FFA, hyperglycemia, and ANG II on the genesis of ROS, COX-2, and PGs and their interplay in the early stages of hyperglcyemia.
Endogenous ephrinB2 mediates colon-urethra cross-organ sensitization via Src kinase-dependent tyrosine phosphorylation of NR2B
Authors: Peng Hsien-Yu , Chen Gin-Den , Lai Cheng-Hung , Tung Kwong-Chung , Chang Junn-Liang , Lin Tzer-Bin
Published: October 2009

Recently, the role of EphB receptor (EphBR) tyrosine kinase and their ephrinB ligands in spinal pain-related neural plasticity has been identified. To test whether Src-family non-receptor tyrosine kinase-dependent glutamatergic N-methyl-d-aspartate receptor (NMDAR) NR2B subunit phosphorylation underlies lumbosacral spinal EphBR activation to mediate cross-organ sensitization between the colon and the urethra, external urethra sphincter electromyogram activity evoked by pelvic nerve stimulation and protein expression in the lumbosacral (L6–S2) dorsal horn were studied before and after intracolonic mustard oil (MO) instillation. We found MO instillation produced colon-urethra reflex sensitization along with an upregulation of endogenous ephrinB2 expression as well as phosphorylation of EphB1/2, Src-family kinase, and NR2B tyrosine residues. Intrathecal immunoglobulin fusion protein of EphB1 and EphB2 as well as PP2 reversed the reflex sensitization and NR2B phosphorylation caused by MO. All these results suggest that EphBR-ephrinB interactions, which provoke Src-family kinase-dependent NMDAR NR2B phosphorylation at the lumbosacral spinal cord level, are involved in cross-organ sensitization, contributing to the development of viscero-visceral referred pain between the bowel and the urethra.
Angiotensin II stimulates trafficking of NHE3, NaPi2, and associated proteins into the proximal tubule microvilli
Authors: Riquier-Brison Anne D. M. , Leong Patrick K. K. , Pihakaski-Maunsbach Kaarina , McDonough Alicia A.
Published: October 2009

Angiotensin II (ANG II) stimulates proximal tubule (PT) sodium and water reabsorption. We showed that treating rats acutely with the angiotensin-converting enzyme inhibitor captopril decreases PT salt and water reabsorption and provokes rapid redistribution of the Na+/H+exchanger isoform 3 (NHE3), Na+/Pi cotransporter 2 (NaPi2), and associated proteins out of the microvilli. The aim of the present study was to determine whether acute ANG II infusion increases the abundance of PT NHE3, NaPi2, and associated proteins in the microvilli available for reabsorbing NaCl. Male Sprague-Dawley rats were infused with a dose of captopril (12 μg/min for 20 min) that increased PT flow rate ∼20% with no change in blood pressure (BP) or glomerular filtration rate (GFR). When ANG II (20 ng·kg−1·min−1for 20 min) was added to the captopril infusate, PT volume flow rate returned to baseline without changing BP or GFR. After captopril, NHE3 was localized to the base of the microvilli and NaPi2 to subapical cytoplasmic vesicles; after 20 min ANG II, both NHE3 and NaPi2 redistributed into the microvilli, assayed by confocal microscopy and density gradient fractionation. Additional PT proteins that redistributed into low-density microvilli-enriched membranes in response to ANG II included myosin VI, DPPIV, NHERF-1, ezrin, megalin, vacuolar H+-ATPase, aminopeptidase N, and clathrin. In summary, in response to 20 min ANG II in the absence of a change in BP or GFR, multiple proteins traffic into the PT brush-border microvilli where they likely contribute to the rapid increase in PT salt and water reabsorption.