bluuebunny/crossref_metadata_2025 huggingface dataset:
Efficacy of Ambruticin Analogs in a Murine Model of Coccidioidomycosis
Authors: Shubitz Lisa F. , Galgiani John N. , Tian Zong-Qiang , Zhong Ziyang , Timmermans Pieter , Katz Leonard
Published: September 2006

Ambruticin S, an antifungal cyclopropyl-pyran acid, showed curative effects against murine coccidioidal infection. Two analogs of this compound with greater in vitro potency were tested against lethal murine
Coccidioides
infection. Both improved the survival of mice over that of controls; one resulted in near-sterilization of infection.
Potent Antiviral Synergy between Monoclonal Antibody and Small-Molecule CCR5 Inhibitors of Human Immunodeficiency Virus Type 1
Authors: Murga Jose D. , Franti Michael , Pevear Daniel C. , Maddon Paul J. , Olson William C.
Published: September 2006

The chemokine receptor CCR5 provides a portal of entry for human immunodeficiency virus type 1 (HIV-1) into susceptible CD4+cells. Both monoclonal antibody (MAb) and small-molecule CCR5 inhibitors have entered human clinical testing, but little is known regarding their potential interactions. We evaluated the interactions between CCR5 MAbs, small-molecule CCR5 antagonists, and inhibitors of HIV-1 gp120, gp41, and reverse transcriptase in vitro. Inhibition data were analyzed for cooperative effects using the combination index (CI) method and stringent statistical criteria. Potent, statistically significant antiviral synergy was observed between the CCR5 MAb PRO 140 and the small-molecule CCR5 antagonists maraviroc (UK-427,857), vicriviroc (SCH-D), and TAK-779. High-level synergy was observed consistently across various assay systems, HIV-1 envelopes, CCR5 target cells, and inhibition levels. CI values ranged from 0.18 to 0.64 and translated into in vitro dose reductions of up to 14-fold. Competition binding studies revealed nonreciprocal patterns of CCR5 binding by MAb and small-molecule CCR5 inhibitors, suggesting that synergy occurs at the level of receptor binding. In addition, both PRO 140 and maraviroc synergized with the chemokine RANTES, a natural ligand for CCR5; however, additive effects were observed for both small-molecule CCR5 antagonists and PRO 140 in combination with other classes of HIV-1 inhibitors. The findings provide a rationale for clinical exploration of MAb and small-molecule CCR5 inhibitors in novel dual-CCR5 regimens for HIV-1 therapy.
Role of P Glycoprotein in Absorption of Novel Antimalarial Drugs
Authors: Crowe Andrew , Ilett Kenneth F. , Karunajeewa Harin A. , Batty Kevin T. , Davis Timothy M. E.
Published: September 2006

Bidirectional transport of four novel antimalarial compounds was determined using Caco-2 cell monolayers. P glycoprotein-mediated efflux was greatest for pyronaridine (5 to 20 μM) and low for naphthoquine (5 μM). With 20 μM naphthoquine, net efflux was blocked, suggesting saturation of the transporter. Piperaquine and dihydroartemisinin were not transported by the system.
System for Expression of Microsporidian Methionine Amino Peptidase Type 2 (MetAP2) in the Yeast
Saccharomyces cerevisiae
Authors: Upadhya Rajendra , Zhang Hong Shan , Weiss Louis M.
Published: September 2006

Microsporidia are parasitic protists of all classes of vertebrates and most invertebrates. They recently emerged as important infections in various immunosuppressed and immunocompetent patient populations. They are also important veterinary and agricultural pathogens. Current therapies for microsporidiosis include benzimidazoles, which bind tubulin-inhibiting microtubule assembly, and fumagillin and its derivatives, which bind and inhibit methionine amino peptidase type 2 (MetAP2). Benzimidazoles are not active against
Enterocytozoon bieneusi
, the most common cause of human microsporidiosis. Fumagillin is active against most microsporidia, including
E. bieneusi
, but thrombocytopenia has been a problem in clinical trials. There is a pressing need for more-specific microsporidian MetAP2 inhibitors. To expedite and facilitate the discovery of safe and effective MetAP2 inhibitors, we have engineered
Saccharomyces cerevisiae
to be dependent on
Encephalitozoon cuniculi
MetAP2 (EcMetAP2) for its growth, where EcMetAP2 is harbored on an episomal uracil-selectable tetracycline-regulated plasmid. We have also constructed a leucine-selectable tetracycline-regulated expression plasmid into which any MetAP2 gene can be cloned. By utilizing a 5-fluoroorotic acid-mediated plasmid shuffle in the EcMetAP2 yeast strain, a yeast strain can be generated whose growth is dependent on MetAP2 from any organism. The level of heterologous MetAP2 gene expression can be controlled by the addition of tetracycline to the growth medium. These yeast strains should permit high-throughput screening for the identification of new inhibitors with high specificity and activity toward microsporidian MetAP2.
In Vitro Susceptibility of Breakthrough
Candida
Bloodstream Isolates Correlates with Daily and Cumulative Doses of Fluconazole
Authors: Clancy Cornelius J. , Staley Benjamin , Nguyen M. Hong
Published: September 2006

We determined MICs for 28
Candida
isolates recovered from patients who developed breakthrough candidemia while receiving fluconazole. MICs correlated with daily and cumulative doses of fluconazole. Patients receiving ≥2,000 mg cumulatively or >200 mg daily were more likely to be infected with isolates that were not fluconazole susceptible.
Comparative In Vitro Efficacies and Antimicrobial Durabilities of Novel Antimicrobial Central Venous Catheters
Authors: Hanna Hend , Bahna Paul , Reitzel Ruth , Dvorak Tanya , Chaiban Gassan , Hachem Ray , Raad Issam
Published: September 2006

We investigated the efficacies and durability of novel antimicrobial central venous catheters (CVCs) in preventing the adherence of microbial organisms to the surfaces of the CVCs. Novel antimicrobial CVCs investigated in this in vitro study were impregnated with antibiotics (minocycline and rifampin), with Oligon agent (silver, platinum, and carbon black), with approved antiseptics (chlorhexidine and silver sulfadiazine), or with a novel antiseptic agent, gendine, which contains gentian violet and chlorhexidine. When tested against methicillin-resistant
Staphylococcus aureus
(MRSA) and
Pseudomonas aeruginosa
, gendine-coated CVC segments provided protection against bacterial adherence significantly more than all other types of tested CVCs (
P
< 0.05). Gendine-coated CVCs also provided better protection against
Candida albicans
and
Candida parapsilosis
than CVCs impregnated with antibiotics or with silver, platinum, and carbon (
P
< 0.02). After 28 days of being soaked in serum, the CVCs impregnated with chlorhexidine and silver sulfadiazine and the CVCs impregnated with silver, platinum, and carbon had lost antimicrobial activity against MRSA,
P. aeruginosa
, and
C. parapsilosis
, and the CVCs impregnated with minocycline and rifampin had lost activity against
P. aeruginosa
and
C. parapsilosis
. The CVCs impregnated with gendine maintained antimicrobial activities against MRSA,
P. aeruginosa
, and
C. parapsilosis
after 28 days of being soaked in serum. Central venous catheters impregnated with the novel investigational antiseptic gendine showed in vitro efficacy and provided protection against bacterial adherence more than other approved novel antimicrobial-coated CVCs.
Murine Model for Dengue Virus-Induced Lethal Disease with IncreasedVascular Permeability
Authors: Shresta Sujan , Sharar Kristin L. , Prigozhin Daniil M. , Beatty P. Robert , Harris Eva
Published: September 2006

Lack
of an appropriate animal model for dengue virus (DEN), which causes
dengue fever and dengue hemorrhagic fever/dengue shock syndrome
(DHF/DSS), has impeded characterization of the mechanisms underlying
the disease pathogenesis. The cardinal feature of DHF/DSS, the severe
form of DEN infection, is increased vascular permeability. To develop a
murine model that is more relevant to DHF/DSS, a novel DEN strain,
D2S10, was generated by alternately passaging a non-mouse-adapted DEN
strain between mosquito cells and mice, thereby mimicking the natural
transmission cycle of the virus between mosquitoes and humans. After
infection with D2S10, mice lacking interferon receptors died early
without manifesting signs of paralysis, carried infectious virus in
both non-neuronal and neuronal tissues, and exhibited signs of
increased vascular permeability. In contrast, mice infected with the
parental DEN strain developed paralysis at late times after infection,
contained detectable levels of virus only in the central nervous
system, and displayed normal vascular permeability. In the mice
infected with D2S10, but not the parental DEN strain, significant
levels of serum tumor necrosis factor alpha (TNF-α) were
produced, and the neutralization of TNF-α activity prevented
early death of D2S10-infected mice. Sequence analysis comparing D2S10
to its parental strain implicated a conserved region of amino acid
residues in the envelope protein as a possible source for the D2S10
phenotype. These results demonstrate that D2S10 causes a more relevant
disease in mice and that TNF-α may be one of several key
mediators of severe DEN-induced disease in mice. This report represents
a significant advance in animal models for severe DEN disease, and it
begins to provide mechanistic insights into DEN-induced disease in
vivo.
Coactivator-Associated Arginine Methyltransferase 1 Enhances Transcriptional Activity ofthe Human T-Cell Lymphotropic Virus Type 1 Long Terminal Repeat through Direct Interactionwith Tax
Authors: Jeong Soo-Jin , Lu Hanxin , Cho Won-Kyung , Park Hyeon Ung , Pise-Masison Cynthia , Brady John N.
Published: September 2006

In this study, we demonstrate that the coactivator-associated arginine
methyltransferase 1 (CARM1), which methylates histone H3 and other
proteins such as p300/CBP, is positively involved in the regulation of
Tax transactivation. First, transfection studies demonstrated that
overexpression of CARM1 wild-type protein resulted in increased Tax
transactivation of the human T-cell lymphotropic virus type 1 (HTLV-1)
long terminal repeat (LTR). In contrast, transfection of a
catalytically inactive CARM1 methyltransferase mutant did not enhance
Tax transactivation. CARM1 facilitated Tax transactivation of the
CREB-dependent cellular GEM promoter. A direct physical interaction
between HTLV-1 Tax and CARM1 was demonstrated using in vitro
glutathione
S
-transferase-Tax binding assays, in vivo
coimmunoprecipitation, and confocal microscopy experiments. Finally,
chromatin immunoprecipitation analysis of the activated HTLV-1 LTR
promoter showed the association of CARM1 and methylated histone H3 with
the template DNA. In vitro, Tax facilitates the binding of CARM1 to the
transcription complex. Together, our data provide evidence that CARM1
enhances Tax transactivation of the HTLV-1 LTR through a direct
interaction between CARM1 and Tax and this binding promotes methylation
of histone H3 (R2, R17, and
R26).
Infection of CD127+(Interleukin-7 Receptor+) CD4+Cells and Overexpression of CTLA-4 Are Linked to Loss of Antigen-Specific CD4 T Cells during Primary Human Immunodeficiency Virus Type 1 Infection
Authors: Zaunders John J. , Ip Susanna , Munier Mee Ling , Kaufmann Daniel E. , Suzuki Kazuo , Brereton Choechoe , Sasson Sarah C. , Seddiki Nabila , Koelsch Kersten , Landay Alan , Grey Pat , Finlayson Robert , Kaldor John , Rosenberg Eric S. , Walker Bruce D. , Fazekas de St. Groth Barbara , Cooper David A. , Kelleher Anthony D.
Published: September 2006

We recently found that human immunodeficiency virus (HIV)-specific CD4+T cells express coreceptor CCR5 and activation antigen CD38 during early primary HIV-1 infection (PHI) but then rapidly disappear from the circulation. This cell loss may be due to susceptibility to infection with HIV-1 but could also be due to inappropriate apoptosis, an expansion of T regulatory cells, trafficking out of the circulation, or dysfunction. We purified CD38+++CD4+T cells from peripheral blood mononuclear cells, measured their level of HIV-1 DNA by PCR, and found that about 10% of this population was infected. However, a small subset of HIV-specific CD4+T cells also expressed CD127, a marker of long-term memory cells. Purified CD127+CD4+lymphocytes contained fivefold more copies of HIV-1 DNA per cell than did CD127-negative CD4+cells, suggesting preferential infection of long-term memory cells. We observed no apoptosis of antigen-specific CD4+T cells in vitro and only a small increase in CD45RO+CD25+CD127dimCD4+T regulatory cells during PHI. However, 40% of CCR5+CD38+++CD4+T cells expressed gut-homing integrins, suggesting trafficking through gut-associated lymphoid tissue (GALT). Furthermore, 80% of HIV-specific CD4+T cells expressed high levels of the negative regulator CTLA-4 in response to antigen stimulation in vitro, which was probably contributing to their inability to produce interleukin-2 and proliferate. Taken together, the loss of HIV-specific CD4+T cells is associated with a combination of an infection of CCR5+CD127+memory CD4+T cells, possibly in GALT, and a high expression of the inhibitory receptor CTLA-4.
Functional Interaction between Human Herpesvirus 6 Immediate-Early 2 Protein and Ubiquitin-Conjugating Enzyme 9 in the Absence of Sumoylation
Authors: Tomoiu Andru , Gravel Annie , Tanguay Robert M. , Flamand Louis
Published: September 2006

The immediate-early 2 (IE2) protein of human herpesvirus 6 is a potent transactivator of cellular and viral promoters. To better understand the biology of IE2, we generated a LexA-IE2 fusion protein and screened, using the yeast two-hybrid system, a Jurkat T-cell cDNA library for proteins that could interact with IE2. The most frequently isolated IE2-interacting protein was the human ubiquitin-conjugating enzyme 9 (Ubc9), a protein involved in the small ubiquitin-like modifier (SUMO) conjugation pathway. Using deletion mutants of IE2, we mapped the IE2-Ubc9-interacting region to residues 989 to 1037 of IE2. The interaction was found to be of functional significance to IE2, as Ubc9 overexpression significantly repressed promoter activation by IE2. The C93S Ubc9 mutant exhibited a similar effect on IE2, indicating that the E2 SUMO-conjugating function of Ubc9 is not required for its repressive action on IE2. No consensus sumoylation sites or evidence of IE2 conjugation to SUMO could be demonstrated under in vivo or in vitro conditions. Moreover, expression levels and nuclear localization of IE2 were not altered by Ubc9 overexpression, suggesting that Ubc9's repressive function likely occurs at the transcriptional complex level. Overall, our results indicate that Ubc9 influences IE2's function and provide new information on the complex interactions that occur between herpesviruses and the sumoylation pathway.
JC Virus Evolution and Its Association with Human Populations
Authors: Shackelton Laura A. , Rambaut Andrew , Pybus Oliver G. , Holmes Edward C.
Published: September 2006

The ubiquitous human polyomavirus JC (JCV) is a small double-stranded DNA virus that establishes a persistent infection, and it is often transmitted from parents to children. There are at least 14 subtypes of the virus associated with different human populations. Because of its presumed codivergence with humans, JCV has been used as a genetic marker for human evolution and migration. Codivergence has also been used as a basis for estimating the rate of nucleotide substitution in JCV. We tested the hypothesis of host-virus codivergence by (i) performing a reconciliation analysis of phylogenetic trees of human and JCV populations and (ii) providing the first estimate of the evolutionary rate of JCV that is independent from the assumption of codivergence. Strikingly, our comparisons of JCV and human phylogenies provided no evidence for codivergence, suggesting that this virus should not be used as a marker for human population history. Further, while the estimated nucleotide substitution rate of JCV has large confidence intervals due to limited sampling, our analysis suggests that this virus may evolve nearly two orders of magnitude faster than predicted under the codivergence hypothesis.
Neutralized Adenovirus-Immune Complexes Can Mediate Effective Gene Transfer via an Fc Receptor-Dependent Infection Pathway
Authors: Leopold Philip L. , Wendland Rebecca L. , Vincent Theresa , Crystal Ronald G.
Published: September 2006

Neutralization of adenovirus (Ad) by anti-Ad neutralizing antibodies in serum involves formation of Ad-immune complexes that prevent the virus from interacting with target cells. We hypothesized that Ad-immune complexes likely contain viable Ad vectors which, although no longer capable of gaining access to receptors on target cells, may be able to express transgenes in cells bearing Fc receptors for immunoglobulins, i.e., that antibody-based “neutralization” of Ad vectors may be circumvented by the Fc receptor pathway. To test this hypothesis, we expressed the Fcγ receptor IIA (FcγR) in A549 lung epithelial cells or human dermal fibroblasts and evaluated gene transfer in the presence of human neutralizing anti-Ad serum. FcγR-expressing cells bound and internalized copious amounts of Ad, with a distinct population of internalized Ad trafficking to the nucleus. The dose-response curves for inhibition of gene transfer revealed that FcγR-expressing cells required a more-than-10-fold higher concentration of anti-Ad serum to achieve 50% inhibition of Ad-encoded β-galactosidase expression compared with non-FcγR-expressing cells. The discrepancy between neutralization of Ad during infection of FcγR-expressing cells and neutralization of Ad during infection of non-FcγR-expressing cells occurred with either heat-inactivated or non-heat-inactivated sera, was blocked by addition of purified Fc domain protein, and did not require the cytoplasmic domain of FcγR, suggesting that immune complex internalization proceeded via endocytosis rather than phagocytosis. FcγR-mediated infection by Ad-immune complexes did not require expression of the coxsackie virus-Ad receptor (CAR) since similar data were obtained when CAR-deficient human dermal fibroblasts were engineered to express FcγR. However, interaction of the Ad penton base with cell surface integrins contributed to the difference in neutralization between FcγR-expressing and non-FcγR-expressing cells. The data indicate that complexes formed from Ad and anti-Ad neutralizing antibodies, while compromised with respect to infection of non-FcγR-expressing target cells, maintain the potential to transfer genes to FcγR-expressing cells, with consequent expression of the transgene. The formation of Ad-immune complexes that can target viable virus to antigen-presenting cells may account for the success of Ad-based vaccines administered in the presence of low levels of neutralizing anti-Ad antibody.
Human T-Cell Responses to Vaccinia Virus Envelope Proteins
Authors: Tang Jie , Murtadha Mariam , Schnell Matthias , Eisenlohr Laurence C. , Hooper Jay , Flomenberg Phyllis
Published: September 2006

One approach for a safer smallpox vaccine is to utilize recombinant subunits rather than live vaccinia virus (VACV). The products of the VACV envelope genes A27L, L1R, B5R, and A33R induce protective antibodies in animal models. We propose that proteins that elicit T-cell responses, as well as neutralizing antibodies, will be important to include in a molecular vaccine. To evaluate VACV-specific memory T-cell responses, peripheral blood mononuclear cells (PBMC) from four VACV vaccinees were tested against whole VACV and the individual envelope proteins A27, B5, L1, and A33, using gamma interferon enzyme-linked immunospot and cytokine flow cytometry assays. PBMC were stimulated with autologous dendritic cells infected with VACV or electroporated with individual VACV protein mRNAs. T-cell lines from all donors, vaccinated from 1 month to over 20 years ago, recognized all four VACV envelope proteins. Both CD4
+
and CD8
+
T-cell responses to each protein were detected. Further analysis focused on representative proteins B5 and A27. PBMC from a recent vaccinee exhibited high frequencies of CD4
+
and CD8
+
T-cell precursors to both B5 (19.8 and 20%, respectively) and A27 (6.8 and 3.7%). In comparison, B5- and A27-specific T-cell frequencies ranged from 0.4 to 1.3% in a donor vaccinated 3 years ago. Multiple CD4
+
and CD8
+
T-cell epitopes were identified from both A27 and B5, using overlapping 15-mer peptides. These data suggest that all four VACV envelope proteins may contribute to protective immunity, not only by inducing antibody responses, but also by eliciting T-cell responses.
Suppression of Proinflammatory Signal Transduction and Gene Expression by the DualNucleic Acid Binding Domains of the Vaccinia Virus E3L Proteins
Authors: Langland Jeffrey O. , Kash John C. , Carter Victoria , Thomas Matthew J. , Katze Michael G. , Jacobs Bertram L.
Published: September 2006

Cells have evolved elaborate mechanisms to counteract the onslaught of viral
infections. To activate these defenses, the viral threat must be
recognized. Danger signals, or pathogen-associated molecular patterns,
that are induced by pathogens include double-stranded RNA (dsRNA),
viral single-stranded RNA, glycolipids, and CpG DNA. Understanding the
signal transduction pathways activated and host gene expression induced
by these danger signals is vital to understanding virus-host
interactions. The vaccinia virus E3L protein is involved in blocking
the host antiviral response and increasing pathogenesis, functions that
map to separate C-terminal dsRNA- and N-terminal Z-DNA-binding domains.
Viruses containing mutations in these domains allow modeling of the
role of dsRNA and Z-form nucleic acid in the host response to virus
infection. Deletions in the Z-DNA- or dsRNA-binding domains led to
activation of signal transduction cascades and up-regulation of host
gene expression, with many genes involved in the inflammatory
response. These data suggest that poxviruses actively
inhibit cellular recognition of viral danger signals and the subsequent
cellular response to the viral
threat.
Differential Enhancement of Dengue Virus Immune Complex Infectivity Mediated by Signaling-Competent and Signaling-Incompetent Human FcγRIA (CD64) or FcγRIIA (CD32)
Authors: Rodrigo W. W. Shanaka I. , Jin Xia , Blackley Shanley D. , Rose Robert C. , Schlesinger Jacob J.
Published: September 2006

Fcγ receptor (FcγR)-mediated entry of infectious dengue virus immune complexes into monocytes/macrophages is hypothesized to be a key event in the pathogenesis of complicated dengue fever. FcγRIA (CD64) and FcγRIIA (CD32), which predominate on the surface of such dengue virus-permissive cells, were compared for their influence on the infectivity of dengue 2 virus immune complexes formed with human dengue virus antibodies. A signaling immunoreceptor tyrosine-based activation motif (ITAM) incorporated into the accessory γ-chain subunit that associates with FcγRIA and constitutively in FcγRIIA is required for phagocytosis mediated by these receptors. To determine whether FcγRIA and FcγRIIA activation functions are also required for internalization of infectious dengue virus immune complexes, we generated native and signaling-incompetent versions of each receptor by site-directed mutagenesis of ITAM tyrosine residues. Plasmids designed to express these receptors were transfected into COS-7 cells, and dengue virus replication was measured by plaque assay and flow cytometry. We found that both receptors mediated enhanced dengue virus immune complex infectivity but that FcγRIIA appeared to do so far more effectively. Abrogation of FcγRIA signaling competency, either by expression without γ-chain or by coexpression with γ-chain mutants, was associated with significant impairment of phagocytosis and of dengue virus immune complex infectivity. Abrogation of FcγRIIA signaling competency was also associated with equally impaired phagocytosis but had no discernible effect on dengue virus immune complex infectivity. These findings point to fundamental differences between FcγRIA and FcγRIIA with respect to their immune-enhancing capabilities and suggest that different mechanisms of dengue virus immune complex internalization may operate between these FcγRs.
Naïve T-Cell Depletion Related to Infection by X4 Human Immunodeficiency Virus Type 1 in Poor Immunological Responders to Highly Active Antiretroviral Therapy
Authors: Delobel Pierre , Nugeyre Marie-Thérèse , Cazabat Michelle , Sandres-Sauné Karine , Pasquier Christophe , Cuzin Lise , Marchou Bruno , Massip Patrice , Cheynier Rémi , Barré-Sinoussi Françoise , Izopet Jacques , Israël Nicole
Published: September 2006

The reasons for poor CD4
+
T-cell recovery in some human immunodeficiency virus (HIV)-infected subjects despite effective highly active antiretroviral therapy (HAART) remain unclear. We recently reported that CXCR4-using (X4) HIV-1 could be gradually selected in cellular reservoirs during sustained HAART. Because of the differential expression of HIV-1 coreceptors CCR5 and CXCR4 on distinct T-cell subsets, the residual replication of R5 and X4 viruses could have different impacts on T-cell homeostasis during immune reconstitution on HAART. We examined this hypothesis and the mechanisms of CD4
+
T-cell restoration by comparing the virological and immunological features of 15 poor and 15 good immunological responders to HAART. We found a high frequency of X4 viruses in the poor immunological responders. But the levels of intrathymic proliferation of the two groups were similar regardless of whether they were infected by R5 or X4 virus. The frequency of recent thymic emigrants in the poor immunological responders was also similar to that found in the good immunological responders, despite their reduced numbers of naïve CD4
+
T cells. Our data, rather, suggest that the naïve T-cell compartment is drained by a high rate of mature naïve cell loss in the periphery due to bystander apoptosis or activation-induced differentiation. X4 viruses could play a role in the depletion of naïve T cells in poor immunological responders to HAART by triggering persistent T-cell activation and bystander apoptosis via gp120-CXCR4 interactions.
Nuclear Import Defect of Human Immunodeficiency Virus Type 1 DNA Flap Mutants Is Not Dependent on the Viral Strain or Target Cell Type
Authors: Arhel Nathalie , Munier Sandie , Souque Philippe , Mollier Karine , Charneau Pierre
Published: September 2006

We have previously established, using human immunodeficiency virus type 1 (HIV-1) strain LAI, that the HIV-1 central DNA Flap acts as acisdeterminant of viral genome nuclear import. Although the impact of the DNA Flap on nuclear import has already found numerous independent confirmations in the context of lentivirus vectors, it has been claimed that it may be nonessential for infectious virus strains LAI, YU-2 (J. D. Dvorin et al., J. Virol.76:12087-12096, 2002), HXB2, and NL4-3 (A. Limon et al., J. Virol.76:12078-12086, 2002). We conducted a detailed analysis of virus infectivity using the provirus clones provided by the authors and analogous target cells. In contrast to published data, our results show that all cPPT mutant viruses exhibit reduced infectivity corresponding to a nuclear import defect irrespective of the viral genetic background or target cell.
Catalytic Core of Alphavirus Nonstructural Protein nsP4 Possesses Terminal Adenylyltransferase Activity
Authors: Tomar Shailly , Hardy Richard W. , Smith Janet L. , Kuhn Richard J.
Published: September 2006

The RNA-dependent RNA polymerase nsP4 is an integral part of the alphavirus replication complex. To define the role of nsP4 in viral RNA replication and for a structure-function analysis, we expressed Sindbis virus nsP4 in
Escherichia coli
. The core catalytic domain of nsP4 (Δ97nsP4, a deletion of the N-terminal 97 amino acids), which consists of the predicted polymerase domain containing the GDD amino acid motif required for viral RNA synthesis, was stable against proteolytic degradation during expression. Therefore, the recombinant core domain and selected mutants were expressed and purified to homogeneity. We determined that Δ97nsP4 possesses terminal adenylyltransferase (TATase) activity, as it specifically catalyzed the addition of adenine to the 3′ end of an acceptor RNA in the presence of divalent cations. Furthermore, Δ97nsP4 is unable to transfer other nucleotides (UTP, CTP, GTP, and dATP) to the acceptor RNA in the absence or presence of other nucleotides. Δ97nsP4 possessing a GDD-to-GAA mutation completely inactivates the enzymatic activity. However, a GDD-to-SNN mutation did not inactivate the enzyme but reduced its activity to ∼45% of that of the wild type in the presence of Mg
2+
. Investigation of the TATase of the GDD-to-SNN mutant revealed that it had TATase equivalent to that of the wild type in the presence of Mn
2+
. Identification of Δ97nsP4 TATase activity suggests a novel function of the alphavirus RNA-dependent RNA polymerase in the maintenance and repair of the poly(A) tail, an element required for replication of the viral genome.
Efficient Mother-to-Child Transfer of Antiretroviral Immunity in the Context of Preclinical Monoclonal Antibody-Based Immunotherapy
Authors: Gros Laurent , Pelegrin Mireia , Plays Marc , Piechaczyk Marc
Published: September 2006

When mice under the age of 5 to 6 days are infected, the FrCas
E
retrovirus induces a neurodegenerative disease leading to death within 1 to 2 months. We have recently reported that transient treatment with a neutralizing monoclonal antibody (MAb) shortly after infection, in addition to an expected immediate decrease in the viral load, also favors the development of a strong protective immune response that persists long after the MAb has been cleared. This observation may have important therapeutic consequences, as it suggests that MAbs might be used, not only as direct neutralizing agents, but also as immunomodulatory agents enabling patients to mount their own antiviral immune responses. We have investigated whether immunoglobulins from mothers who displayed a strong anti-FrCas
E
humoral response induced upon MAb treatment could affect both viremia and the immune systems of FrCas
E
-infected pups till adult age upon placental and/or breastfeeding transfer. The strongest effects, i.e., reduction in the viral load and induction of protective humoral antiviral responses, were observed upon breastfeeding alone and breastfeeding plus placental immunity transfer. However, placental transfer of anti-FrCas
E
antibodies was sufficient to both protect neonatally infected animals and help them initiate a neutralizing anti-FrCas
E
response. Also, administration of a neutralizing MAb to naive mothers during late gestation and breastfeeding could generate similar effects. Taken together, our data support the concept that passive immunotherapies during late gestation and/or breastfeeding might help retrovirally infected neonates prime their own protective immune responses, in addition to exerting an immediate antiviral effect.
Induction of a Protective Immune Response against Viral Nervous Necrosis in the European Sea Bass
Dicentrarchus labrax
by Using Betanodavirus Virus-Like Particles
Authors: Thiéry R. , Cozien J. , Cabon J. , Lamour F. , Baud M. , Schneemann A.
Published: September 2006

Betanodaviruses are causative agents of viral nervous necrosis (VNN), a devastating disease of cultured marine fish worldwide. Virus particles contain a single type of coat protein that spontaneously assembles into virus-like particles (VLPs) when expressed in a baculovirus expression system. In the present study, the immunogenicity of betanodavirus VLPs and the protection they confer against VNN in the European sea bass
Dicentrarchus labrax
were investigated. Enzyme-linked immunosorbent assay and seroneutralization tests performed on plasma from fish vaccinated intramuscularly with doses as low as 0.1 μg of VLPs indicated that the VLPs elicited the synthesis of specific antibetanodavirus antibodies with neutralizing activity. Moreover, fish vaccinated with VLPs were protected from challenge with live virus. Both the immune response and the protective effect against viral challenge were dose dependent. Reverse transcription-PCR data indicated that higher doses of vaccine also reduced the number of fish containing detectable quantities of betanodavirus RNA on day 30 after challenge. Taken together these data strongly support the hypothesis that VLPs obtained in the baculovirus expression system may represent an effective vaccine against VNN.
Asynchronous Progression through the Lytic Cascade and Variations in Intracellular Viral Loads Revealed by High-Throughput Single-Cell Analysis of Kaposi's Sarcoma-Associated Herpesvirus Infection
Authors: Adang Laura A. , Parsons Christopher H. , Kedes Dean H.
Published: September 2006

Kaposi's sarcoma-associated herpesvirus (KSHV or human herpesvirus-8) is frequently tumorigenic in immunocompromised patients. The average intracellular viral copy number within infected cells, however, varies markedly by tumor type. Since the KSHV-encoded latency-associated nuclear antigen (LANA) tethers viral episomes to host heterochromatin and displays a punctate pattern by fluorescence microscopy, we investigated whether accurate quantification of individual LANA dots is predictive of intracellular viral genome load. Using a novel technology that integrates single-cell imaging with flow cytometry, we found that both the number and the summed immunofluorescence of individual LANA dots are directly proportional to the amount of intracellular viral DNA. Moreover, combining viral (immediate early lytic replication and transcription activator [RTA] and late lytic K8.1) and cellular (syndecan-1) staining with image-based flow cytometry, we were also able to rapidly and simultaneously distinguish among cells supporting latent, immediate early lytic, early lytic, late lytic, and a potential fourth “delayed late” category of lytic replication. Applying image-based flow cytometry to KSHV culture models, we found that de novo infection results in highly varied levels of intracellular viral load and that lytic induction of latently infected cells likewise leads to a heterogeneous population at various stages of reactivation. These findings additionally underscore the potential advantages of studying KSHV biology with high-throughput analysis of individual cells.
Tyro3 Family-Mediated Cell Entry of Ebola and Marburg Viruses
Authors: Shimojima Masayuki , Takada Ayato , Ebihara Hideki , Neumann Gabriele , Fujioka Kouki , Irimura Tatsuro , Jones Steven , Feldmann Heinz , Kawaoka Yoshihiro
Published: September 2006

Filoviruses, represented by the genera
Ebolavirus
and
Marburgvirus
, cause a lethal hemorrhagic fever in humans and in nonhuman primates. Although filovirus can replicate in various tissues or cell types in these animals, the molecular mechanisms of its broad tropism remain poorly understood. Here we show the involvement of members of the Tyro3 receptor tyrosine kinase family—Axl, Dtk, and Mer—in cell entry of filoviruses. Ectopic expression of these family members in lymphoid cells, which otherwise are highly resistant to filovirus infection, enhanced infection by pseudotype viruses carrying filovirus glycoproteins on their envelopes. This enhancement was reduced by antibodies to Tyro3 family members, Gas6 ligand, or soluble ectodomains of the members. Live Ebola viruses infected both Axl- and Dtk-expressing cells more efficiently than control cells. Antibody to Axl inhibited infection of pseudotype viruses in a number of Axl-positive cell lines. These results implicate each Tyro3 family member as a cell entry factor in filovirus infection.
COMMUNICATION COMPLEXITY AND SPEED-UP IN THE EXPLICIT DIFFERENCE METHOD
Authors: PURCZ PAVOL
Published: September 2006

An earlier suggested parallel "ring" algorithm for solving the spatially one-dimensional initial-boundary-value problem (IBVP) for a parabolic equation using an explicit difference method is shortly described. Theoretical estimates for both of the speed-up function and the communication complexity of this parallel algorithm are studied. The speed-up function is determined as the ratio between times for realization of the algorithm in sequentional and parallel cases. Theoretical estimates of the speed-up function show the significant speed-up of the parallel algorithm in comparison with the serial one for a large number of values computed by one processor during one time level and it is shown that the the speed-up tends to the number of used processors. Communication complexity is determined as a ratio between the number of interchanges and the number of arithmetical operations. It has been proven that the coefficient of the communication complexity for spatially m-dimensional IBVP tends in general to ¾.
COMMUNICATION PERFORMANCE OF LAM/MPI AND MPICH ON A LINUX CLUSTER
Authors: ROZMAN IGOR , ŠTERK MARJAN , TROBEC ROMAN
Published: September 2006

High performance parallel computers provide computational rates necessary for computer simulations and intensive computing applications. An important part of a parallel computer program is an MPI software library, which implements communication within parallel applications. Several MPI implementations exist, most widely used among them are LAM/MPI and MPICH. This paper presents results of four basic synthetic tests and two real simulations in LAM/MPI and MPICH environments. Tests were made on a computer cluster composed of 17 dual-processor nodes connected by a toroidal mesh. Results show that on the investigated cluster, LAM outperformed MPICH especially by bidirectional ring communication, and that appropriate trimming of communication parameters significantly contributes to the final parallel performance.
SUPERSYMMETRIC LORENTZ INVARIANT DEFORMATIONS OF SUPERSPACES
Authors: ZHELTUKHIN A. A.
Published: September 2006

Lorentz invariant supersymmetric deformations of superspaces based on Moyal star product parametrized by Majorana spinor λa and Ramond Grassmannian vector [Formula: see text] in the spinor realization42 are proposed. The map of supergravity background into composite supercoordinates: [Formula: see text] valid up to the second order corrections in deformation parameter h and transforming the background dependent Lorentz noninvariant (anti)commutators of supercoordinates into their invariant Moyal brackets is revealed. We found one of the deformations to depend on the axial vector [Formula: see text] and to vanish for the θ components with the same chiralities. The deformations in the (super)twistor picture are discussed.
MULTIPLICITY OF POSITIVE SOLUTIONS FOR A SINGULAR AND CRITICAL ELLIPTIC PROBLEM IN ℝ2
Authors: ADIMURTHI  , GIACOMONI JACQUES
Published: September 2006

In this paper, we are interested in the following singular problem: [Formula: see text] where 0 < p ∈ L∞, λ > 0, 0 < δ < 3, Ω a smooth bounded domain. We show on suitable conditions on h there exist two solutions in [Formula: see text]. Investigating the radial case, we are able to prove that the condition at infinity for h is optimal for getting the existence of the second solution.
STRUCTURES, ABSORPTION SPECTRA, AND ELECTRONIC PROPERTIES OF POLYFLUORENE AND ITS DERIVATIVES: A THEORETICAL STUDY
Authors: SRIWICHITKAMOL KRIENGSAK , SURAMITR SONGWUT , POOLMEE POTJAMAN , HANNONGBUA SUPA
Published: September 2006

The structural and energetic properties of polyfluorene and its derivatives were investigated, using quantum chemical calculations. Conformational analysis of bifluorene was performed by using ab initio (HF/6-31G* and MP2/6-31G*) and density functional theory (B3LYP/6-31G*) calculations. The results showed that the local energy minimum of bifluorene lies between the coplanar and perpendicular conformation, and the B3LYP/6-31G* calculations led to the overestimation of the stability of the planar pi systems. The HOMO-LUMO energy differences of fluorene oligomers and its derivatives — 9,9-dihexylfluorene (DHPF), 9,9-dioctylfluorene (PFO), and bis(2-ethylhexyl)fluorene (BEHPF) — were calculated at the B3LYP/6-31G* level. Energy gaps and effective conjugation lengths of the corresponding polymers were obtained by extrapolating HOMO-LUMO energy differences and the lowest excitation energies to infinite chain length. The lowest excitation energies and the maximum absorption wavelength of polyfluorene were also performed, employing the time-dependent density functional theory (TDDFT) and ZINDO methods. The extrapolations, based on TDDFT and ZINDO calculations, agree well with experimental results. These theoretical methods can be useful for the design of new polymeric structures with a reducing energy gap.
STUDY OF PROTON TRANSFER BETWEEN CARBON ACIDS AND AMINE BASES USING COMPUTATIONAL METHODS
Authors: JALILI SEIFOLLAH , SOLEIMANI MINA
Published: September 2006

In this work, the proton transfer between carbon acids and amine bases was investigated using computational methods. The effect of substitutions on the interaction between 1-nitro-1-(4-nitrophenyl) alkanes (with R = H , CH 3) and 1-5-7-triazabicyclo[4,4,0]dec-5-ene) (TBD) was investigated. The solvent effects on proton transfer reaction between 1-nitro-1-(4-nitrophenyl) alkanes (in which R = H , CH 3) and TBD have been calculated in acetonitrile and water solvents. The products of proton transfer reactions between C -acids and 7-methyl-1-5-7-triazabicyclo[4,4,0]dec-5-ene (MTBD) were dissociated into free ions, while those of the TBD reaction contained a comparable amount of ions and ion pairs. In addition, the free energy of activation for proton transfer reaction between C -acids with R = H , CH 3 and TBD was calculated. The intermolecular hydrogen bonding formation between 4-nitrophenylnitromethane and TBD was investigated using the Hartree–Fock method, density functional theory (DFT), and atoms in molecules (AIM) theory. The effects of hydrogen bonding on structural parameters of model systems were also studied. The results are in agreement with the results of AIM theory.
Effects of streptozotocin-induced diabetes and physical training on gene expression of titin-based stretch-sensing complexes in mouse striated muscle
Authors: Lehti T. Maarit , Silvennoinen Mika , Kivelä Riikka , Kainulainen Heikki , Komulainen Jyrki
Published: September 2006

In striated muscle, a sarcomeric noncontractile protein, titin, is proposed to form the backbone of the stress- and strain-sensing structures. We investigated the effects of diabetes, physical training, and their combination on the gene expression of proteins of putative titin stretch-sensing complexes in skeletal and cardiac muscle. Mice were divided into control (C), training (T), streptozotocin-induced diabetic (D), and diabetic training (DT) groups. Training groups performed for 1, 3, or 5 wk of endurance training on a motor-driven treadmill. Muscle samples from T and DT groups together with respective controls were collected 24 h after the last training session. Gene expression of calf muscles (soleus, gastrocnemius, and plantaris) and cardiac muscle were analyzed using microarray and quantitative PCR. Diabetes induced changes in mRNA expression of the proteins of titin stretch-sensing complexes in Z-disc (MLP, myostatin), I-band (CARP, Ankrd2), and M-line (titin kinase signaling). Training alleviated diabetes-induced changes in most affected mRNA levels in skeletal muscle but only one change in cardiac muscle. In conclusion, we showed diabetes-induced changes in mRNA levels of several fiber-type-biased proteins (MLP, myostatin, Ankrd2) in skeletal muscle. These results are consistent with previous observations of diabetes-induced atrophy leading to slower fiber type composition. The ability of exercise to alleviate diabetes-induced changes may indicate slower transition of fiber type.
Acute ethanol exposure in pregnancy alters the insulin-like growth factor axis of fetal and maternal sheep
Authors: Gatford Kathryn L. , Dalitz Penelope A. , Cock Megan L. , Harding Richard , Owens Julie A.
Published: September 2006

Maternal ethanol intake during pregnancy impairs fetal growth, but mechanisms are not clearly defined. Reduced IGF abundance or bioavailability in the fetus and/or mother may contribute to this growth restriction. We hypothesized that an episode of acute ethanol exposure, mimicking binge drinking would restrict fetal growth and perturb the maternal and fetal IGF axes. Pregnant sheep were infused intravenously with saline or ethanol (1 g/kg maternal wt) over 1 h, on days 116, 117, and 118 of gestation (start of 1st infusion = time 0, term is 147 days). Maternal and fetal plasma IGF and IGF-binding protein (IGFBP) concentrations were measured before and after each infusion. Compared with controls, ethanol exposure reduced fetal weight at day 120 by 19%, transiently reduced maternal plasma IGF-I (−35%) at 30 h, and decreased fetal plasma IGF-II (−28%) from 24 to 54 h after the first infusion. Ethanol exposure did not alter maternal or fetal plasma concentrations of IGFBP-2 and IGFBP-3, measured by Western ligand blotting. We conclude that suppression of maternal and fetal IGF abundance may contribute to fetal growth restriction induced by acute or binge ethanol exposure.
Expression and localization of androgen receptor-interacting protein-4 in the testis
Authors: Domanskyi Andrii , Zhang Fu-Ping , Nurmio Mirja , Palvimo Jorma J. , Toppari Jorma , Jänne Olli A.
Published: September 2006

Androgen receptor-interacting protein 4 (ARIP4) belongs to the SNF2 family of proteins involved in chromatin remodeling, DNA excision repair, and homologous recombination. It is a DNA-dependent ATPase, binds to DNA and mononucleosomes, and interacts with androgen receptor (AR) and modulates AR-dependent transactivation. We have examined in this study the expression and cellular localization of ARIP4 during postnatal development of mouse testis. ARIP4 was detected by immunohistochemistry in Sertoli cell nuclei at all ages studied, starting on day 5, and exhibited the highest expression level in adult mice. At the onset of spermatogenesis, ARIP4 expression became evident in spermatogonia, pachytene, and diplotene spermatocytes. Immunoreactive ARIP4 antigen was present in Leydig cell nuclei. In Sertoli cells ARIP4 was expressed in a stage-dependent manner, with high expression levels at stages II–VI and VII–VIII. ARIP4 expression patterns did not differ significantly in testes of wild-type, follicle-stimulating hormone receptor knockout, and luteinizing hormone receptor knockout mice. In testes of hypogonadal mice, ARIP4 was found mainly in interstitial cells and exhibited lower expression in Sertoli and germ cells. In vitro stimulation of rat seminiferous tubule segments with testosterone, FSH, or forskolin did not significantly change stage-specific levels of ARIP4 mRNA. Heterozygous ARIP4+/− mice were haploinsufficient and had reduced levels of Sertoli-cell specific androgen-regulated Rhox5 (also called Pem) mRNA. Collectively, ARIP4 is an AR coregulator in Sertoli cells in vivo, but the expression in the germ cells implies that it has also AR-independent functions in spermatogenesis.