bluuebunny/crossref_metadata_2025 huggingface dataset:
Dihydroxythiophenes Are Novel Potent Inhibitors of Human Immunodeficiency Virus Integrase with a Diketo Acid-Like Pharmacophore
Authors: Kehlenbeck S. , Betz U. , Birkmann A. , Fast B. , Göller A. H. , Henninger K. , Lowinger T. , Marrero D. , Paessens A. , Paulsen D. , Pevzner V. , Schohe-Loop R. , Tsujishita H. , Welker R. , Kreuter J. , Rübsamen-Waigmann H. , Dittmer F.
Published: June 2006

We have identified dihydroxythiophenes (DHT) as a novel series of human immunodeficiency virus type 1 (HIV-1) integrase inhibitors with broad antiviral activities against different HIV isolates in vitro. DHT were discovered in a biochemical integrase high-throughput screen searching for inhibitors of the strand transfer reaction of HIV-1 integrase. DHT are selective inhibitors of integrase that do not interfere with virus entry, as shown by the inhibition of a vesicular stomatitis virus G-pseudotyped retroviral system. Moreover, in quantitative real-time PCR experiments, no effect on the synthesis of viral cDNA could be detected but rather an increase in the accumulation of 2-long-terminal-repeat cycles was detected. This suggests that the integration of viral cDNA is blocked. Molecular modeling and the structure activity relationship of DHT demonstrate that our compound fits into a two-metal-binding motif that has been suggested as the essential pharmacophore for diketo acid (DKA)-like strand transfer inhibitors (Grobler et al., Proc. Natl. Acad. Sci. USA 99:6661-6666, 2002.). This notion is supported by the profiling of DHT on retroviral vectors carrying published resistance mutations for DKA-like inhibitors where DHT showed partial cross-resistance. This suggests that DHT bind to a common site in the catalytic center of integrase, albeit with an altered binding mode. Taken together, our findings indicate that DHT are novel selective strand transfer inhibitors of integrase with a pharmacophore homologous to DKA-like inhibitors.
Hypersusceptibility to Substrate Analogs Conferred by Mutations in Human Immunodeficiency Virus Type 1 Reverse Transcriptase
Authors: Smith Robert A. , Anderson Donovan J. , Preston Bradley D.
Published: June 2006

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) contains four structural motifs (A, B, C, and D) that are conserved in polymerases from diverse organisms. Motif B interacts with the incoming nucleotide, the template strand, and key active-site residues from other motifs, suggesting that motif B is an important determinant of substrate specificity. To examine the functional role of this region, we performed “random scanning mutagenesis” of 11 motif B residues and screened replication-competent mutants for altered substrate analog sensitivity in culture. Single amino acid replacements throughout the targeted region conferred resistance to lamivudine and/or hypersusceptibility to zidovudine (AZT). Substitutions at residue Q151 increased the sensitivity of HIV-1 to multiple nucleoside analogs, and a subset of these Q151 variants was also hypersusceptible to the pyrophosphate analog phosphonoformic acid (PFA). Other AZT-hypersusceptible mutants were resistant to PFA and are therefore phenotypically similar to PFA-resistant variants selected in vitro and in infected patients. Collectively, these data show that specific amino acid replacements in motif B confer broad-spectrum hypersusceptibility to substrate analog inhibitors. Our results suggest that motif B influences RT-deoxynucleoside triphosphate interactions at multiple steps in the catalytic cycle of polymerization.
ICP0 Gene Expression Is a Herpes Simplex Virus Type 1 Apoptotic Trigger
Authors: Sanfilippo Christine M. , Blaho John A.
Published: June 2006

Apoptosis is a highly regulated programmed cell death process which is activated during normal development and by various stimuli, such as viral infection, which disturb cellular metabolism and physiology. That herpes simplex virus type 1 (HSV-1) induces apoptosis but then prevents its killing of infected cells is well-established. However, little is known about the viral factor/event which triggers the apoptotic process. We previously reported that infections with either (i) a temperature-sensitive virus at its nonpermissive temperature which does not inject viral DNA into nuclei or (ii) various UV-inactivated wild-type viruses do not result in the induction of apoptosis (C. M. Sanfilippo, F. N. W. Chirimuuta, and J. A. Blaho, J. Virol. 78:224-239, 2004). This indicates that virus receptor binding/attachment to cells, membrane fusion, virion disassembly/tegument dispersal, virion RNAs, and capsid translocation to nuclei are not responsible for induction and implicates viral immediate-early (IE) gene expression in the process. Here, we systematically evaluated the contribution of each IE gene to the stimulation of apoptosis. Using a series of viruses individually deleted for α27, α4, and α22, we determined that these genes are not required for apoptosis induction but rather that their products play roles in its prevention, likely through regulatory effects. Sole expression of α0 acted as an “apoptoxin” that was necessary and sufficient to trigger the cell death cascade. Importantly, results using a recombinant virus which contains a stop codon in α0 showed that it was not the ICP0 protein which acted as the apoptotic inducer. Based on these findings, we propose that α0 gene expression acts as an initial inducer of apoptosis during HSV-1 infection. This represents the first description of apoptosis induction in infected cells triggered as a result of expression of a single viral gene. Expression of apoptotic viral genes is a unique mechanism through which human pathogens may modulate interactions with their host cells.
Evaluating Replication-Defective Vesicular Stomatitis Virus as a Vaccine Vehicle
Authors: Majid Ayaz M. , Ezelle Heather , Shah Sangeeta , Barber Glen N.
Published: June 2006

We have generated replication-competent (VSV-C/E1/E2) and nonpropagating (VSVΔG-C/E1/E2) vesicular stomatitis virus (VSV) contiguously expressing the structural proteins of hepatitis C virus (HCV; core [C] and glycoproteins E1 and E2) and report on their immunogenicity in murine models. VSV-C/E1/E2 and VSVΔG-C/E1/E2 expressed high levels of HCV C, E1, and E2, which were authentically posttranslationally processed. Both VSV-expressed HCV E1-E2 glycoproteins were found to form noncovalently linked heterodimers and appeared to be correctly folded, as confirmed by coimmunoprecipitation analysis using conformationally sensitive anti-HCV-E2 monoclonal antibodies (MAbs). Intravenous or intraperitoneal immunization of BALB/c mice with VSV-C/E1/E2 or VSVΔG-C/E1/E2 resulted in significant and surprisingly comparable HCV core or E2 antibody responses compared to those of control mice. In addition, both virus types generated HCV C-, E1-, or E2-specific gamma interferon (IFN-γ)-producing CD8
+
T cells, as determined by enzyme-linked immunospot (ELISPOT) analysis. Mice immunized with VSVΔG-C/E1/E2 were also protected against the formation of tumors expressing HCV E2 (CT26-hghE2t) and exhibited CT26-hghE2t-specific IFN-γ-producing and E2-specific CD8
+
T-cell activity. Finally, recombinant vaccinia virus (vvHCV.S) expressing the HCV structural proteins replicated at significantly lower levels when inoculated into mice immunized with VSV-C/E1/E2 or VSVΔG-C/E1/E2, but not with control viruses. Our data therefore illustrate that potentially safer replication-defective VSV can be successfully engineered to express high levels of antigenically authentic HCV glycoproteins. In addition, this strategy may therefore serve in effective vaccine and immunotherapy-based approaches to the treatment of HCV-related disease.
The 2.6-Angstrom Structure of Infectious Bursal Disease Virus-Derived T=1 Particles Reveals New Stabilizing Elements of the Virus Capsid
Authors: Garriga Damià , Querol-Audí Jordi , Abaitua Fernando , Saugar Irene , Pous Joan , Verdaguer Núria , Castón José R. , Rodriguez José F.
Published: June 2006

Infectious
bursal disease virus (IBDV), a member of the
Birnaviridae
family, is a double-stranded RNA virus that causes a highly contagious
disease in young chickens leading to significant economic losses in the
poultry industry. The VP2 protein, the only structural component of the
IBDV icosahedral capsid, spontaneously assembles into T=1 subviral
particles (SVP) when individually expressed as a chimeric gene. We have
determined the crystal structure of the T=1 SVP to 2.60 Å
resolution. Our results show that the 20 trimeric VP2 clusters forming
the T=1 shell are further stabilized by calcium ions located at the
threefold icosahedral axes. The structure also reveals a new unexpected
domain swapping that mediates interactions between adjacent trimers: a
short helical segment located close to the end of the long C-terminal
arm of VP2 is projected toward the threefold axis of a neighboring VP2
trimer, leading to a complex network of interactions that increases the
stability of the T=1 particles. Analysis of crystal packing shows that
the exposed capsid residues, His253 and Thr284, determinants of IBDV
virulence and the adaptation of the virus to grow in cell culture, are
involved in particle-particle
interactions.
Both Spike and Background Genes Contribute to Murine Coronavirus Neurovirulence
Authors: Iacono Kathryn T. , Kazi Lubna , Weiss Susan R.
Published: June 2006

Various strains of mouse hepatitis virus (MHV) exhibit different pathogenic phenotypes. Infection with the A59 strain of MHV induces both encephalitis and hepatitis, while the highly neurovirulent JHM strain induces a fatal encephalitis with little, if any, hepatitis. The pathogenic phenotype for each strain is determined by the genetic composition of the viral genome, as well as the host immune response. Using isogenic recombinant viruses with A59 background genes differing only in the spike gene, we have previously shown that high neurovirulence is associated with the JHM spike protein, the protein responsible for attachment to the host cell receptor (J. J. Phillips, M. M. Chua, G. F. Rall, and S. R. Weiss, Virology301:109-120, 2002). Using another set of isogenic recombinant viruses with JHM background genes expressing either the JHM or A59 spike, we have further investigated the roles of viral genes in pathogenesis. Here, we demonstrate that the high neurovirulence of JHM is associated with accelerated spread through the brain and a heightened innate immune response that is characterized by high numbers of infiltrating neutrophils and macrophages, suggesting an immunopathogenic component to neurovirulence. While expression of the JHM spike is sufficient to confer a neurovirulent phenotype, as well as increased macrophage infiltration, background genes contribute to virulence as well, at least in part, by dictating the extent of the T-cell immune response.
Epstein-Barr Virus LMP2A Enhances B-Cell Responses In Vivo and In Vitro
Authors: Swanson-Mungerson Michelle , Bultema Rebecca , Longnecker Richard
Published: June 2006

Epstein-Barr virus (EBV) establishes latent infections in a significant percentage of the population. Latent membrane protein 2A (LMP2A) is an EBV protein expressed during latency that inhibits B-cell receptor signaling in lymphoblastoid cell lines. In the present study, we have utilized a transgenic mouse system in which LMP2A is expressed in B cells that are specific for hen egg lysozyme (E/HEL-Tg). To determine if LMP2A allows B cells to respond to antigen, E/HEL-Tg mice were immunized with hen egg lysozyme. E/HEL-Tg mice produced antibody in response to antigen, indicating that LMP2A allows B cells to respond to antigen. In addition, E/HEL-Tg mice produced more antibody and an increased percentage of plasma cells after immunization compared to HEL-Tg littermates, suggesting that LMP2A increased the antibody response in vivo. Finally, in vitro studies determined that LMP2A acts directly on the B cell to increase antibody production by augmenting the expansion and survival of the activated B cells, as well as increasing the percentage of plasma cells generated. Taken together, these data suggest that LMP2A enhances, not diminishes, B-cell-specific antibody responses in vivo and in vitro in the E/HEL-Tg system.
Vaccinia Virus Activation of CCR5 Invokes Tyrosine Phosphorylation Signaling Events That Support Virus Replication
Authors: Rahbar Ramtin , Murooka Thomas T. , Hinek Anna A. , Galligan Carole L. , Sassano Antonella , Yu Celeste , Srivastava Kishore , Platanias Leonidas C. , Fish Eleanor N.
Published: June 2006

Vaccinia virus, a poxvirus, produces structurally distinct forms of virions for which the immediate events following cell entry are ill-defined. We provide evidence that intracellular mature virus (IMV) enters both permissive and nonpermissive T-cell lines and that introduction of CCR5 into nonpermissive mouse fibroblasts or human primary T cells renders the cells permissive for vaccinia replication. Notably, T cells expressing CCR5 in which tyrosine 339 in the intracellular region is replaced by phenylalanine no longer support virus replication or virus-inducible activation of specific host cell signaling effectors IRS-2, Grb2, and Erk1/2. We show that following IMV entry into the cell, the intact but not the tyrosine-deficient CCR5 is rapidly internalized and colocalizes with virus. This colocalization precedes virus-inducible signaling and replication.
Isolation and Characterization of Human Monoclonal Antibodies from Individuals Infected with West Nile Virus
Authors: Throsby Mark , Geuijen Cecile , Goudsmit Jaap , Bakker Arjen Q. , Korimbocus Jehanara , Kramer R. Arjen , Clijsters-van der Horst Marieke , de Jong Maureen , Jongeneelen Mandy , Thijsse Sandra , Smit Renate , Visser Therese J. , Bijl Nora , Marissen Wilfred E. , Loeb Mark , Kelvin David J. , Preiser Wolfgang , ter Meulen Jan , de Kruif John
Published: June 2006

Monoclonal antibodies (MAbs) neutralizing West Nile Virus (WNV) have been shown to protect against infection in animal models and have been identified as a correlate of protection in WNV vaccine studies. In the present study, antibody repertoires from three convalescent WNV-infected patients were cloned into an scFv phage library, and 138 human MAbs binding to WNV were identified. One hundred twenty-one MAbs specifically bound to the viral envelope (E) protein and four MAbs to the premembrane (prM) protein. Enzyme-linked immunosorbent assay-based competitive-binding assays with representative E protein-specific MAbs demonstrated that 24/51 (47%) bound to domain II while only 4/51 (8%) targeted domain III. In vitro neutralizing activity was demonstrated for 12 MAbs, and two of these, CR4374 and CR4353, protected mice from lethal WNV challenge at 50% protective doses of 12.9 and 357 μg/kg of body weight, respectively. Our data analyzing three infected individuals suggest that the human anti-WNV repertoire after natural infection is dominated by nonneutralizing or weakly neutralizing MAbs binding to domain II of the E protein, while domain III-binding MAbs able to potently neutralize WNV in vitro and in vivo are rare.
Genetic Organization and Hypoxic Activation of the Kaposi's Sarcoma-Associated Herpesvirus ORF34-37 Gene Cluster
Authors: Haque Muzammel , Wang Victoria , Davis David A. , Zheng Zhi-Ming , Yarchoan Robert
Published: June 2006

Kaposi's
sarcoma-associated herpesvirus (KSHV) is the etiologic agent for
Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). We
previously reported that hypoxia activates KSHV lytic replication and
that the promoter for open reading frame 34 (ORF34) contains a
functional hypoxia-responsive element (HRE). ORF34 is part of a cluster
of lytic genes (ORF34-37) that includes ORF36, a phosphotransferase,
and ORF37, a shutoff exonuclease. Rapid amplification of cDNA ends
analysis revealed that they share a common polyadenylation signal but
have two start sites. Two transcripts were identified, one 3.4 kb
encoding ORF35-37, and the other 4.2 kb encoding ORF34 and also having
coding potential for ORF35-37. Exposure of PEL cell lines to hypoxia
induced messages of lengths consistent with those of these transcripts.
Reporter assays with Hep3B cells showed activation of both transcripts
by hypoxia. The ORF34-37 promoter region has six consensus HREs.
Sequential deletion, site-directed mutagenesis experiments, and
Northern blot analysis of RNA produced by constructs indicated that the
second HRE (HRE-2) plays a critical role in the hypoxic activation of
both RNA transcripts. The ORF35-37 transcript was upregulated by
cotransfected hypoxia-inducible factor (HIF). Electrophoretic mobility
shift assays demonstrated that HRE-2 and ancillary sequences bind and
compete for HIF with hypoxic Hep3B nuclear extract. The activation of
this gene cluster by hypoxia may have implications for the pathogenesis
of PEL and KS. Moreover, the activation of ORF36 by hypoxia might be
exploited to develop targeted therapy for PEL, which arises in a
hypoxic environment (pleural
effusions).
Localization of ERK/MAP Kinase Is Regulated by the Alphaherpesvirus Tegument Protein Us2
Authors: Lyman Mathew G. , Randall Jessica A. , Calton Christine M. , Banfield Bruce W.
Published: June 2006

Many different viruses activate the extracellular signal-regulated kinase (ERK)/mitogen-activated protein (MAP) kinase signaling pathway during infection and require ERK activation for the efficient execution of their replication programs. Despite these findings, no virus-encoded proteins have been identified that directly modulate ERK activities. In an effort to determine the function of a conserved alphaherpesvirus structural protein called Us2, we screened a yeast two-hybrid library derived from NIH 3T3 cells and identified ERK as a Us2-interacting protein. Our studies indicate that Us2 binds to ERK in virus-infected cells, mediates the incorporation of ERK into the virion, and inhibits the activation of ERK nuclear substrates. The association of Us2 with ERK leads to the sequestration of ERK at the plasma membrane and to a perinuclear vesicular compartment, thereby keeping ERK out of the nucleus. Us2 can bind to activated ERK, and the data suggest that Us2 does not inhibit ERK enzymatic activity. The treatment of cells with U0126, a specific inhibitor of ERK activation, resulted in a substantial delay in the release of virus from infected cells that was more pronounced with a virus deleted for Us2 than with parental and repaired strains, suggesting that both ERK and Us2 activities are required for efficient virus replication. This study highlights an additional complexity to the activation of ERK by viruses, namely, that localization of active ERK can be altered by virus-encoded proteins.
Protein-Primed and De Novo Initiation of RNA Synthesis by Norovirus 3D
pol
Authors: Rohayem Jacques , Robel Ivonne , Jäger Katrin , Scheffler Ulrike , Rudolph Wolfram
Published: June 2006

Noroviruses (
Caliciviridae
) are RNA viruses with a single-stranded, positive-oriented polyadenylated genome. To date, little is known about the replication strategy of norovirus, a so-far noncultivable virus. We have examined the initiation of replication of the norovirus genome in vitro, using the active norovirus RNA-dependent RNA polymerase (3D
pol
), homopolymeric templates, and synthetic subgenomic or antisubgenomic RNA. Initiation of RNA synthesis on homopolymeric templates as well as replication of subgenomic polyadenylated RNA was strictly primer dependent. In this context and as observed for other enteric RNA viruses, i.e., poliovirus, a protein-primed initiation of RNA synthesis after elongation of the VPg by norovirus 3D
pol
was postulated. To address this question, norovirus VPg was expressed in
Escherichia coli
and purified. Incubation of VPg with norovirus 3D
pol
generated VPg-poly(U), which primed the replication of subgenomic polyadenylated RNA. In contrast, replication of antisubgenomic RNA was not primer dependent, nor did it depend on a leader sequence, as evidenced by deletion analysis of the 3′ termini of subgenomic and antisubgenomic RNA. On nonpolyadenylated RNA, i.e., antisubgenomic RNA, norovirus 3D
pol
initiated RNA synthesis de novo and terminated RNA synthesis by a poly(C) stretch. Interestingly, on poly(C) RNA templates, norovirus 3D
pol
initiated RNA synthesis de novo in the presence of high concentrations of GTP. We propose a novel model for initiation of replication of the norovirus genome by 3D
pol
, with a VPg-protein-primed initiation of replication of polyadenylated genomic RNA and a de novo initiation of replication of antigenomic RNA.
Modest but Reproducible Inhibition of Human Immunodeficiency Virus Type 1 Infection in Macrophages following LEDGFp75 Silencing
Authors: Zielske Steven P. , Stevenson Mario
Published: June 2006

LEDGFp75 is a cellular protein which binds human immunodeficiency virus type 1 (HIV-1) integrase with high specificity and affinity but whose function in infection has not been defined. We infected LEDGFp75-deficient primary macrophages with wild-type HIV in order to assess potential infection phenotypes which would provide clues to LEDGFp75 function. Silencing of LEDGFp75 by 70 to 80% resulted in an average of 53% reduced infection of macrophages by HIV. Analysis of infection intermediates showed that integration, but not two-long-terminal-repeat (2LTR) circles or late cDNAs, was reduced up to 74% in LEDGFp75-deficient macrophages. Therefore, LEDGFp75 has a modest involvement in HIV-1 integration in macrophages.
The Capsid Protein of Beak and Feather Disease Virus Binds to the Viral DNA and Is Responsible for Transporting the Replication-Associated Protein into the Nucleus
Authors: Heath Livio , Williamson Anna-Lise , Rybicki Edward P.
Published: June 2006

Circoviruses lack an autonomous DNA polymerase and are dependent on the replication machinery of the host cell for de novo DNA synthesis. Accordingly, the viral DNA needs to cross both the plasma membrane and the nuclear envelope before replication can occur. Here we report on the subcellular distribution of the beak and feather disease virus (BFDV) capsid protein (CP) and replication-associated protein (Rep) expressed via recombinant baculoviruses in an insect cell system and test the hypothesis that the CP is responsible for transporting the viral genome, as well as Rep, across the nuclear envelope. The intracellular localization of the BFDV CP was found to be directed by three partially overlapping bipartite nuclear localization signals (NLSs) situated between residues 16 and 56 at the N terminus of the protein. Moreover, a DNA binding region was also mapped to the N terminus of the protein and falls within the region containing the three putative NLSs. The ability of CP to bind DNA, coupled with the karyophilic nature of this protein, strongly suggests that it may be responsible for nuclear targeting of the viral genome. Interestingly, whereas Rep expressed on its own in insect cells is restricted to the cytoplasm, coexpression with CP alters the subcellular localization of Rep to the nucleus, strongly suggesting that an interaction with CP facilitates movement of Rep into the nucleus.
Factors Determining the Breadth and Potency of Neutralization by V3-Specific Human Monoclonal Antibodies Derived from Subjects Infected with Clade A or Clade B Strains of Human Immunodeficiency Virus Type 1
Authors: Krachmarov C. P. , Honnen W. J. , Kayman S. C. , Gorny M. K. , Zolla-Pazner S. , Pinter Abraham
Published: June 2006

The neutralizing activities of anti-V3 antibodies for HIV-1 isolates is affected both by sequence variation within V3 and by epitope masking by the V1/V2 domain. To analyze the relative contribution of V3 sequence variation, chimeric Env genes that contained consensus V3 sequences from seven HIV-1 subtypes in the neutralization-sensitive SF162 Env backbone were constructed. Resulting viral pseudotypes were tested for neutralization by 15 anti-V3 MAbs isolated from humans infected with viruses of either subtype B (anti-V3
B
MAbs) or subtype A (anti-V3
A
MAbs). Pseudovirions with the subtype B consensus V3 sequence were potently neutralized (IC
50
< 0.006 μg/ml) by all but one of these MAbs, while pseudovirions with V3 subtypes A, C, F, H, AG, and AE were generally neutralized more effectively by anti-V3
A
MAbs than by anti-V3
B
MAbs. A V1/V2-masked Env version of SF162 Env with the consensus B V3 sequence was also neutralized by these MAbs, although with considerably lower potency, while similarly masked chimeras with V3 sequences of subtype A, C, or AG were weakly neutralized by anti-V3
A
MAbs but not by anti-V3
B
MAbs. Mutations in the V1/V2 domain of YU-2 Env increased the sensitivity of this highly resistant Env to a pool of anti-V3
B
MAbs several thousand-fold. These results demonstrated (i) the exceptional sensitivity of representative V3 domains of multiple subtypes to neutralization in the absence of epitope masking, (ii) the broader neutralizing activity of anti-V3
A
MAbs for viruses containing diverse V3 sequences, and (iii) the generality and dominant effect of V1/V2 masking on restriction of V3-mediated neutralization.
CD1d Mediates T-Cell-Dependent Resistance to Secondary Infection with Encephalomyocarditis Virus (EMCV) In Vitro and Immune Response to EMCV Infection In Vivo
Authors: Ilyinskii Petr O. , Wang Ruojie , Balk Steven P. , Exley Mark A.
Published: June 2006

The innate and adaptive immune responses have evolved distinct strategies for controlling different viral pathogens. Encephalomyocarditis virus (EMCV) is a picornavirus that can cause paralysis, diabetes, and myocarditis within days of infection. The optimal innate immune response against EMCV in vivo requires CD1d. Interaction of antigen-presenting cell CD1d with distinct natural killer T-cell (“NKT”) populations can induce rapid gamma interferon (IFN-γ) production and NK-cell activation. The T-cell response of CD1d-deficient mice (lacking all NKT cells) against acute EMCV infection was further studied in vitro and in vivo. EMCV persisted at higher levels in CD1d-knockout (KO) splenocyte cultures infected in vitro. Furthermore, optimal resistance to repeat cycles of EMCV infection in vitro was also shown to depend on CD1d. However, this was not reflected in the relative levels of NK-cell activation but rather by the responses of both CD4+and CD8+T-cell populations. Repeated EMCV infection in vitro induced less IFN-γ and alpha interferon (IFN-α) from CD1d-deficient splenocytes than with the wild type. Furthermore, the level of EMCV replication in wild-type splenocytes was markedly and specifically increased by addition of blocking anti-CD1d antibody. Depletion experiments demonstrated that dendritic cells contributed less than the combination of NK and NKT cells to anti-EMCV responses and that none of these cell types was the main source of IFN-α. Finally, EMCV infection in vivo produced higher levels of viremia in CD1d-KO mice than in wild-type animals, coupled with significantly less lymphocyte activation and IFN-α production. These results point to the existence of a previously unrecognized mechanism of rapid CD1d-dependent stimulation of the antiviral adaptive cellular immune response.
Fitness Comparison of Thymidine Analog Resistance Pathways in Human Immunodeficiency Virus Type 1
Authors: Hu Zixin , Giguel Françoise , Hatano Hiroyu , Reid Patrick , Lu Jing , Kuritzkes Daniel R.
Published: June 2006

Resistance to zidovudine (ZDV) results from thymidine analog resistance mutations (TAMs) at human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) codons 41, 67, 70, 210, 215, and 219. Two mutations are possible at codon 215: Y or F. Whereas T215Y occurs alone or with M41L and L210W (TAM-1 pattern), T215F rarely occurs with these mutations or by itself; it is found instead with D67N, K70R, and K219Q (TAM-2 pattern). The L210W mutation most often occurs with M41L and T215Y and rarely occurs with the T215F or TAM-2 mutation. To explain these associations, TAMs were introduced into HIV-1Hxb2by site-directed mutagenesis and expressed in recombinant viruses. Viral replication kinetics, relative fitness, and infectivity were tested in the absence or presence of ZDV. Viruses carrying the 215Y mutation showed faster replication kinetics and greater relative fitness than did T215F mutants in the absence or presence of ZDV. In addition, T215Y mutants showed greater infectivity than did wild-type HIV-1 over a range of ZDV concentrations, but T215F mutants had only a modest advantage over the wild-type virus. Whereas introduction of L210W improved the relative fitness of an M41L/T215Y mutant in the presence of ZDV, introduction of this mutation into a D67N/K70R/K219Q background resulted in decreased relative fitness in the presence or absence of drug. By contrast, introduction of T215F into the D67N/K70R/K219Q background increased viral fitness in the presence of ZDV. These results help explain why T215Y but not T215F usually emerges as the first major TAM, as well as the clustering of L210W with TAM-1 mutations and T215F with TAM-2 mutations.
Physeal injuries in children’s and youth sports: reasons for concern?
Authors: Caine D , DiFiori J , Maffulli N
Published: June 2006

A systematic review of the literature on the frequency and characteristics of sports related growth plate injuries affecting children and youth in organised sport was carried out. Both acute and chronic physeal injuries related to participation in sports have been reported to occur, although injury incidence data are somewhat limited. Of particular concern is the growing number of stress related physeal injuries, including those affecting the lower extremities. Although most physeal injuries appeared to resolve with treatment and rest, there is also evidence of growth disturbance and deformity. Possible injury risk factors and countermeasures are discussed, and suggestions for directing future research provided.
Ultrasound guided electrocoagulation in patients with chronic non-insertional Achilles tendinopathy: a pilot study
Authors: Boesen M Ilum , Torp-Pedersen S , Koenig M Juhl , Christensen R , Langberg H , Hölmich P , Nielsen M Bachmann , Bliddal H
Published: June 2006

Background:High resolution colour Doppler ultrasound shows intratendinous Doppler activity in patients with chronic Achilles tendinopathy. Treatment of this neovascularisation with sclerosing therapy seems to relieve the pain. However, the procedure often has to be repeated.

Objective:To investigate the effect of electrocoagulation of the neovessels on tendon pain and tendon vascularity in patients with chronic Achilles tendinopathy.

Methods:Colour Doppler ultrasound guided electrocoagulation was used on vessels in the ventral portion of the Achilles tendon in 11 patients (seven men, four women, mean age 41 years) with painful chronic mid-portion Achilles tendinosis. A unipolar coagulation device was used.

Results:One patient dropped out after two months (dissatisfied with the results). The remaining 10 patients (91%) were satisfied. These 10 patients were still satisfied at six months of follow up and had returned to their previous level of activity. All 10 patients were “cured” after one treatment. The patient who dropped out received two treatments because of lack of progress. There was significantly reduced pain (Likert pain scale, 0–10) during activity, from a median of 7 (range 4 to 10) at baseline to 0 (0 to 8) at six months’ follow up (p<0.005); and at rest, from 1.5 (1 to 5) to 0 (0 to 8) (p = 0.005). In all patients, vascularisation was unchanged at the six months follow up, with no significant change in semiquantitative or quantitative colour scoring.

Conclusions:Coagulation in the area with vessels entering the tendon appears to be effective treatment for painful chronic mid-tendinous Achilles tendinopathy. No effect on the intratendinous Doppler activity could be detected, suggesting that the effect is independent of changes in blood flow. Localisation of hyperaemia appears to be the key to the pathology and for targeting the treatment. One explanation could be that the effect is obtained by destruction of nerves accompanying the vessels.
Mobilisation of mesenchymal cells into blood in response to skeletal muscle injury
Authors: Ramírez M , Lucia A , Gómez-Gallego F , Esteve-Lanao J , Pérez-Martínez A , Foster C , Andreu A L , Martin M A , Madero L , Arenas J , García-Castro J
Published: June 2006

Mesenchymal cells recruited to damaged tissues must circulate through the bloodstream. The absolute numbers of circulating mesenchymal stem cells (cMSCs) in two different models of acute and chronic skeletal muscle injury were determined. cMSCs were present in significantly higher numbers in both models than in healthy controls. These results support the hypothesis that MSCs are mobilised into the bloodstream after skeletal muscle tissue damage. These two models (acute and chronic) would be of value in the search for molecular mediators of mobilisation of MSCs into the circulation.
Physiological diversity of mitochondrial oxidative phosphorylation
Authors: Benard G. , Faustin B. , Passerieux E. , Galinier A. , Rocher C. , Bellance N. , Delage J.-P. , Casteilla L. , Letellier T. , Rossignol R.
Published: June 2006

To investigate the physiological diversity in the regulation and control of mitochondrial oxidative phosphorylation, we determined the composition and functional features of the respiratory chain in muscle, heart, liver, kidney, and brain. First, we observed important variations in mitochondrial content and infrastructure via electron micrographs of the different tissue sections. Analyses of respiratory chain enzyme content by Western blot also showed large differences between tissues, in good correlation with the expression level of mitochondrial transcription factor A and the activity of citrate synthase. On the isolated mitochondria, we observed a conserved molar ratio between the respiratory chain complexes and a variable stoichiometry for coenzyme Q and cytochrome c, with typical values of [1–1.5]:[30–135]:[3]:[9–35]:[6.5–7.5] for complex II:coenzyme Q:complex III:cytochrome c:complex IV in the different tissues. The functional analysis revealed important differences in maximal velocities of respiratory chain complexes, with higher values in heart. However, calculation of the catalytic constants showed that brain contained the more active enzyme complexes. Hence, our study demonstrates that, in tissues, oxidative phosphorylation capacity is highly variable and diverse, as determined by different combinations of 1) the mitochondrial content, 2) the amount of respiratory chain complexes, and 3) their intrinsic activity. In all tissues, there was a large excess of enzyme capacity and intermediate substrate concentration, compared with what is required for state 3 respiration. To conclude, we submitted our data to a principal component analysis that revealed three groups of tissues: muscle and heart, brain, and liver and kidney.
Mitoenergetic failure in Alzheimer disease
Authors: Parihar Mordhwaj S , Brewer Gregory J.
Published: June 2006

Brain cells are highly energy dependent for maintaining ion homeostasis during high metabolic activity. During active periods, full mitochondrial function is essential to generate ATP from electrons that originate with the oxidation of NADH. Decreasing brain metabolism is a significant cause of cognitive abnormalities of Alzheimer disease (AD), but it remains uncertain whether this is the cause of further pathology or whether synaptic loss results in a lower energy demand. Synapses are the first to show pathological symptoms in AD before the onset of clinical symptoms. Because synaptic function has high energy demands, interruption in mitochondrial energy supply could be the major factor in synaptic failure in AD. A newly discovered age-related decline in neuronal NADH and redox ratio may jeopardize this function. Mitochondrial dehydrogenases and several mutations affecting energy transfer are frequently altered in aging and AD. Thus, with the accumulation of genetic defects in mitochondria at the level of energy transfer, the issue of neuronal susceptibility to damage as a function of age and age-related disease becomes important. In an aging rat neuron model, mitochondria are both chronically depolarized and produce more reactive oxygen species with age. These concepts suggest that multiple treatment targets may be needed to reverse this multifactorial disease. This review summarizes new insights based on the interaction of mitoenergetic failure, glutamate excitotoxicity, and amyloid toxicity in the exacerbation of AD.
Lidocaine depresses splenocyte immune functions following trauma-hemorrhage in mice
Authors: Kawasaki Takashi , Choudhry Mashkoor A. , Schwacha Martin G. , Bland Kirby I. , Chaudry Irshad H.
Published: June 2006

Traumatic and/or surgical injury as well as hemorrhage induces profound suppression of cellular immunity. Although local anesthetics have been shown to impair immune responses, it remains unclear whether lidocaine affects lymphocyte functions following trauma-hemorrhage (T-H). We hypothesized that lidocaine will potentiate the suppression of lymphocyte functions after T-H. To test this, we randomly assigned male C3H/HeN (6–8 wk) mice to sham operation or T-H. T-H was induced by midline laparotomy and ∼90 min of hemorrhagic shock (blood pressure 35 mmHg), followed by fluid resuscitation (4× shed blood volume in the form of Ringer lactate). Two hours later, the mice were killed and splenocytes and bone marrow cells were isolated. The effects of lidocaine on concanavalin A-stimulated splenocyte proliferation and cytokine production in both sham-operated and T-H mice were assessed. The effects of lidocaine on LPS-stimulated bone marrow cell proliferation and cytokine production were also assessed. The results indicate that T-H suppresses cell proliferation, Th1 cytokine production, and MAPK activation in splenocytes. In contrast, cell proliferation, cytokine production, and MAPK activation in bone marrow cells were significantly higher 2 h after T-H compared with shams. Lidocaine depressed immune responses in splenocytes; however, it had no effect in bone marrow cells in either sham or T-H mice. The enhanced immunosuppressive effects of lidocaine could contribute to the host's enhanced susceptibility to infection following T-H.
Inhibitory Postsynaptic Potentials in Lumbar Motoneurons Remain Depolarizing After Neonatal Spinal Cord Transection in the Rat
Authors: Jean-Xavier Céline , Pflieger Jean-François , Liabeuf Sylvie , Vinay Laurent
Published: June 2006

GABA and glycine are excitatory in the immature spinal cord and become inhibitory during development. The shift from depolarizing to hyperpolarizing inhibitory postsynaptic potentials (IPSPs) occurs during the perinatal period in the rat, a time window during which the projections from the brain stem reach the lumbar enlargement. In this study, we investigated the effects of suppressing influences of the brain on lumbar motoneurons during this critical period for the negative shift of the reversal potential of IPSPs ( EIPSP). The spinal cord was transected at the thoracic level on the day of birth [postnatal day 0 (P0)]. EIPSP, at P4–P7, was significantly more depolarized in cord-transected than in cord-intact animals ( EIPSPabove and below resting potential, respectively). EIPSPat P4–P7 in cord-transected animals was close to EIPSPat P0–P2. K-Cl cotransporter KCC2 immunohistochemistry revealed a developmental increase of staining in the area of lumbar motoneurons between P0 and P7 in cord-intact animals; this increase was not observed after spinal cord transection. The motoneurons recorded from cord-transected animals were less sensitive to the experimental manipulations aimed at testing the functionality of the KCC2 system, which is sensitive to [K+]oand blocked by bumetanide. Although bumetanide significantly depolarized EIPSP, the shift was less pronounced than in cord-intact animals. In addition, a reduction of [K+]oaffected EIPSPsignificantly only in cord-intact animals. Therefore influences from the brain stem may play an essential role in the maturation of inhibitory synaptic transmission, possibly by upregulating KCC2 and its functionality.
Ionic Mechanism Underlying Recovery of Rhythmic Activity in Adult Isolated Neurons
Authors: Haedo Rodolfo J. , Golowasch Jorge
Published: June 2006

Neurons exhibit long-term excitability changes necessary for maintaining proper cell and network activity in response to various inputs and perturbations. For instance, the adult crustacean pyloric network can spontaneously recover rhythmic activity after complete shutdown resulting from permanent removal of neuromodulatory inputs. Dissociated lobster stomatogastric ganglion (STG) neurons have been shown to spontaneously develop oscillatory activity via excitability changes. Rhythmic electrical stimulation can eliminate these oscillatory patterns in some cells. The ionic mechanisms underlying these changes are only partially understood. We used dissociated crab STG neurons to study the ionic mechanisms underlying spontaneous recovery of rhythmic activity and stimulation-induced activity changes. Similar to lobster neurons, rhythmic activity spontaneously develops in crab STG neurons. Rhythmic hyperpolarizing stimulation can eliminate, but more commonly accelerate, the emergence of stable oscillatory activity depending on Ca2+influx at hyperpolarized voltages. Our main finding is that upregulation of a Ca2+current and downregulation of a high-threshold K+current underlies the spontaneous homeostatic development of oscillatory activity. However, because of a nonlinear dependence on stimulus frequency, hyperpolarization-induced oscillations appear to be inconsistent with a homeostatic regulation of activity. We find no difference in the activity patterns or the underlying ionic currents involved between neurons of the fast pyloric and the slow gastric mill networks during the first 10 days in isolation. Dynamic-clamp experiments confirm that these conductance modifications can explain the observed activity changes. We conclude that spontaneous and stimulation-induced excitability changes in STG neurons can both result in intrinsic oscillatory activity via regulation of the same two conductances.
Vibrissa Myoclonus (Rhythmic Retractions) Driven by Resonance of Excitatory Networks in Motor Cortex
Authors: Castro-Alamancos Manuel A.
Published: June 2006

Rodents use rhythmic vibrissae movements to sense the environment. It is currently unclear whether intrinsic activity in the vibrissa motor cortex (vMI) is capable of driving vibrissa movements on a cycle-by-cycle basis. Disinhibition of vMI results in the occurrence of spontaneous 5- to 15-Hz synchronized oscillations. In behaving rats, this synchronous resonance of vMI is shown here to drive contralateral vibrissa movements that are phase-locked to each cycle of the oscillation. In contrast to active whisking during sensing, which consists of active protractions, the vibrissa movements produced by vMI oscillations consisted of rhythmic retractions. The results demonstrate that rhythmic motor cortex output is capable of driving vibrissa movements on a cycle-by-cycle basis. Such motor output may be primarily expressed during abnormal states such as those related to cortical myoclonous, tremors, and cortical seizures.
Thermal Behavior of Aromatic Polysulfones with Pendent Viologen Groups
Authors: Avram Ecaterina , Lisa Gabriela , Hurduc Natalia
Published: June 2006

Polysulfones with pendent diquaternary 4,4-bipyridinium groups are polysulfones containing pendent viologen groups. They were obtained by the reaction of chloromethylated polysulfones with N-monoquaternary-4,4′-bipyridinium bromides as: N-( p-H, NO2, Cl) phenacyl - 4,4′- bipyridinium bromides and N-carbomethoxymethyl/ethy l-4,4′-bipyridinium bromides (1a-e). The thermal stabilities of polysulfone with pendent viologen groups (2a-e) were investigated. The influence of the chemical structure of pendent viologen groups on the thermal stability was investigated by thermogravimetric analysis in the dynamic conditions of temperature and atmospheric air. The substituted compounds underwent a thermal degradation process that took place by a complex mechanism in three steps. These steps are discussed from points of view of both the kinetic parameters and the thermogravimetric characteristics. The thermostability of low molar mass compounds 1a-e decreased in following order: 1c < 1a < 1e < 1d <1b and the same thermostability order was found in the case of chloromethylated polysulfone derivatives 2a-e (2c < 2a < 2e < 2d < 2b).
Preparation and Properties of Polyamide Dendrons-Poly(4-Vinylpyridine) Complexes via Multiple Hydrogen Bonding
Authors: Jikei Mitsutoshi , Kouketsu Takayuki , Kakimoto Masa-Aki , Yamane Yuji , Ando Isao
Published: June 2006

The preparation and properties of the multiple hydrogen-bonded complexes between polyamide dendrons and poly(4-vinylpyridine) (P4VP) are described. Polyamide dendrons with or without carboxyl groups were prepared as components for the complex with P4VP. A hydrogen-bonded G1-GA (polyamide dendron having five carboxyl groups)/P4VP 1/5 (molar ratio) complex was prepared by mixing G1-GA with P4VP in methanol, followed by removing the solvent. The formation of the hydrogen bond was confirmed by IR measurements. A glass transition temperature ( Tg) of the complex was observed at 179′C by DSC measurement, whereas the melting point of G1-GA (156′C) and Tg of P4VP (144′C) disappeared. The Tg of G1-GA/P4VP complexes was dependent on the molar ratio ([Acceptor]/[Donor]). The maximum Tg at 184′C was observed in the acceptor-rich region. 1H NMR spin-lattice relaxation time ( T1) and diffusion coefficient ( D) were measured to evaluate the mobility of G1-GA or P4VP in solution. Both T1 and D values suggest that the mobility of the dendron and P4VP in the complex is restricted by the formation of multiple hydrogen bondings in solution.
Synthesis and Properties of Carboxylated Dendritic Polyamide-b-Poly(4-vinylpyridine) Block Copolymers Capable of Multiple Hydrogen Bonding
Authors: Jikei Mitsutoshi , Itoh Hideaki , Kouketsu Takayuki , Hayakawa Teruaki , Kakimoto Masa-Aki
Published: June 2006

A dendritic aromatic polyamide having carboxyl groups on the periphery and a benzyl chloride group at the core (Cl-G1-4COOH) was prepared as an initiator for atom transfer radical polymerization (ATRP). The ATRP of 4-vinylpyridine initiated by Cl-G1-4COOH was carried out in the presence of tris[2-(dimethylamino)ethyl]amine (Me6 TREN) and CuCl to form tadpole-shaped dendritic-linear copolymers (P4VP-G1-4COOH). The structure of P4VP-G1-4COOH was confirmed by nuclear magnetic resonance (NMR) and infrared measurements. The thermal and solution properties of P4VP-G1-4COOH are influenced by the multiple hydrogen bonding between the carboxyl groups on the periphery and vinylpyridine segments. A glass transition temperature value that is higher than that of Cl-G1-4COOH could be due to the contribution of the multiple hydrogen bonding. The decreased solubility in methanol and chloroform suggests the presence of the multiple hydrogen bonding. The solubility of each component also affected the association of P4VP-G1-4COOH in solution. A structural model for the molecular interaction of P4VP-G1-4COOH in methanol, chloroform and N, N-dimethylformamide based on the hydrodynamic radii and NMR measurements is proposed..
Promises Made to Me
Authors: Thomson S. Bruce
Published: July 2006

Universities across the globe have marketed their institutions in nondomestic markets to tap into the lucrative international student market. Higher education is big business, but I ask, “At what cost?” International students face many hurdles when studying away from home. Lack of friends, family, and cultural support is the first step that leads to a deep sense of isolation in many students. Most of these students do find friends and a support system, but for a few, the final price is a burden too heavy to bear. This poem was written after 10 months of my own experience as an international student and talking to other students about their concerns.