bluuebunny/crossref_metadata_2025_split huggingface dataset:
Optimization of Feline Immunodeficiency Virus Vectors for RNA Interference
Authors: Harper Scott Q. , Staber Patrick D. , Beck Christine R. , Fineberg Sarah K. , Stein Colleen , Ochoa Dalyz , Davidson Beverly L. 
Published: September 2006

ABSTRACT
RNA interference (RNAi) occurs naturally in plant and animal cells as a means for modulating gene expression. This process has been experimentally manipulated to achieve targeted gene silencing in cells, tissues, and animals, using a variety of vector systems. Here, we tested the hypothesis that vectors based on feline immunodeficiency virus (FIV) could be used for coexpression of reporter constructs and RNAi expression cassettes. We found, unexpectedly, in our initial constructs that placement of RNAi expression cassettes downstream from a polymerase II (pol II)-expressed reporter gene inhibited reporter expression but not vector titer. Through a series of intermediate vector constructs, we found that placement of the RNAi expression cassette relative to the Rev response element and the pol II expression cassette was critical for efficient RNAi and reporter gene expression. These results suggested that steric factors, including RNA structure and recruitment of competing transcriptional machinery, may affect gene expression from FIV vectors. In a second series of studies, we show that target sequence silencing can be achieved in cells transduced by FIV vectors coexpressing reporter genes and 3′ untranslated region resident microRNAs. The optimized FIV-based RNAi expression vectors will find broad use given the extensive tropism of pseudotyped FIV vectors for many cell types in vitro and in vivo.
Evaluation of the Lytic Origins of Replication of Kaposi's Sarcoma-Associated Virus/Human Herpesvirus 8 in the Context of the Viral Genome
Authors: Xu Yiyang , Rodriguez-Huete Alicia , Pari Gregory S. 
Published: September 2006

ABSTRACT
The lytic origins of DNA replication for human herpesvirus 8 (HHV8), oriLyt-L and oriLyt-R, are located between open reading frames K4.2 and K5 and ORF69 and vFLIP, respectively. These lytic origins were elucidated using a transient replication assay. Although this assay is a powerful tool for identifying many herpesvirus lytic origins, it is limited in its ability to evaluate the activity of replication origins in the context of the viral genome. To this end, we investigated the ability of a recombinant HHV8 bacterial artificial chromosome (BAC) to replicate in the absence of oriLyt-R, oriLyt-L, or both oriLyt regions. We generated the HHV8 BAC recombinants (BAC36-ΔOri-R, BAC36-ΔOri-L, and BAC36-ΔOri-RL), which removed one or all of the identified lytic origins. An evaluation of these recombinant BACs revealed that oriLyt-L was sufficient to propagate the viral genome, whereas oriLyt-R alone failed to direct the amplification of viral DNA.
Major Histocompatibility Complex Class II Molecules Promote Human Immunodeficiency Virus Type 1 Assembly and Budding to Late Endosomal/Multivesicular Body Compartments
Authors: Finzi Andrés , Brunet Alexandre , Xiao Yong , Thibodeau Jacques , Cohen Éric A. 
Published: September 2006

ABSTRACTHuman immunodeficiency virus type 1 (HIV-1) assembly, budding, and release occur mostly at the plasma membrane in T lymphocytes as well as in established nonlymphoid cell lines, while in macrophages these processes occur primarily in intracellular compartments that harbor late endosomal/multivesicular body (LE/MVB) markers, including human leukocyte antigen DR (HLA-DR). Major histocompatibility complex class II molecules (MHC-II), which are expressed in macrophages and activated T cells, have been previously reported to induce the formation of multilaminar and multivesicular endocytic MHC-II-like structures analogous to MVB upon their expression in HEK 293 cells. Here, we have examined the role of MHC-II in HIV-1 Gag targeting as well as in virus assembly and release. Expression of HLA-DR in nonlymphoid cell lines induced a relocation of Gag to intracellular compartments that harbored LE/MVB markers and increased the accumulation of viral particles assembling intracellularly. Consequently, viral production and release from the cell surface was found to be substantially decreased in HLA-DR-expressing cells. This process was specific, since it was not observed with HLA-DR molecules lacking their cytoplasmic tails, nor with structurally related but functionally distinct MHC-II molecules such as HLA-DM or HLA-DO. Importantly, virus released intracellularly in HLA-DR-expressing cells retained infectivity. Overall, these results suggest a role of MHC-II molecules in promoting HIV-1 assembly and budding to LE/MVB and raise the possibility that this activity might be part of a normal pathway of virus production in cell types physiologically expressing MHC-II molecules, such as macrophages.
ICP22 Is Required for Wild-Type Composition and Infectivity of Herpes Simplex Virus Type 1 Virions
Authors: Orlando Joseph S. , Balliet John W. , Kushnir Anna S. , Astor Todd L. , Kosz-Vnenchak Magdalena , Rice Stephen A. , Knipe David M. , Schaffer Priscilla A. 
Published: September 2006

ABSTRACTThe immediate-early regulatory protein ICP22 is required for efficient replication of herpes simplex virus type 1 in some cell types (permissive) but not in others (restrictive). In mice infected via the ocular route, the pathogenesis of an ICP22−virus, 22/n199, was altered relative to that of wild-type virus. Specifically, tear film titers of 22/n199-infected mice were significantly reduced at 3 h postinfection relative to those of mice infected with wild-type virus. Further, 22/n199 virus titers were below the level of detection in trigeminal ganglia (TG) during the first 9 days postinfection. On day 30 postinfection, TG from 22/n199-infected mice contained reduced viral genome loads and exhibited reduced expression of latency-associated transcripts and reduced reactivation efficiency relative to TG from wild-type virus-infected mice. Notably, the first detectable alteration in the pathogenesis of 22/n199 in these tests occurred in the eye prior to the onset of nascent virus production. Thus, ICP22−virions appeared to be degraded, cleared, or adsorbed more rapidly than wild-type virions, implying potential differences in the composition of the two virion types. Analysis of the protein composition of purified extracellular virions indicated that ICP22 is not a virion component and that 22/n199 virions sediment at a reduced density relative to wild-type virions. Although similar to wild-type virions morphologically, 22/n199 virions contain reduced amounts of two γ2late proteins, US11 and gC, and increased amounts of two immediate-early proteins, ICP0 and ICP4, as well as protein species not detected in wild-type virions. Although ICP22−viruses replicate to near-wild-type levels in permissive cells, the virions produced in these cells are biochemically and physically different from wild-type virions. These virion-specific differences in ICP22−viruses add a new level of complexity to the functional analysis of this immediate-early viral regulatory protein.
Invasion of Host Cells by JC Virus Identifies a Novel Role for Caveolae in EndosomalSorting of Noncaveolar Ligands
Authors: Querbes W. , O'Hara B. A. , Williams G. , Atwood W. J. 
Published: September 2006

ABSTRACT
Invasion
of glial cells by the human polyomavirus, JC virus (JCV), leads to a
rapidly progressing and uniformly fatal demyelinating disease known as
progressive multifocal leukoencephalopathy. The endocytic trafficking
steps used by JCV to invade cells and initiate infection are not known.
We demonstrated that JCV infection was inhibited by dominant defective
and constitutively active Rab5-GTPase mutants that acted at distinct
steps in endosomal sorting. We also found that labeled JCV colocalized
with labeled cholera toxin B and with caveolin-1 (cav-1) on early
endosomes following internalization by clathrin-dependent endocytosis.
JCV entry and infection were both inhibited by dominant defective
mutants of eps15 and Rab5-GTPase. Expression of a dominant-negative
scaffolding mutant of cav-1 did not inhibit entry or infection by JCV.
A single-cell knockdown experiment using cav-1 shRNA did not inhibit
JCV entry but interfered with a downstream trafficking event important
for infection. These data show that JCV enters cells by
clathrin-dependent endocytosis, is transported immediately to early
endosomes, and is then sorted to a caveolin-1-positive endosomal
compartment. This latter step is dependent on Rab5-GTPase, cholesterol,
caveolin-1, and pH. This is the first example of a ligand that enters
cells by clathrin-dependent endocytosis and is then sorted from early
endosomes to caveosomes, indicating that caveolae-derived vesicles play
a more important role than previously realized in sorting cargo from
early
endosomes.
Vaccinia Virus F9 Virion Membrane Protein Is Required for Entry but Not Virus Assembly, in Contrast to the Related L1 Protein
Authors: Brown Erica , Senkevich Tatiana G. , Moss Bernard 
Published: September 2006

ABSTRACT
All sequenced poxviruses encode orthologs of the vaccinia virus L1 and F9 proteins, which are structurally similar and share about 20% amino acid identity. We found that F9 further resembles L1 as both proteins are membrane components of the mature virion with similar topologies and induce neutralizing antibodies. In addition, a recombinant vaccinia virus that inducibly expresses F9, like a previously described L1 mutant, had a conditional-lethal phenotype: plaque formation and replication of infectious virus were dependent on added inducer. However, only immature virus particles are made when L1 is repressed, whereas normal-looking intracellular and extracellular virions formed in the absence of F9. Except for the lack of F9, the polypeptide components of such virions were indistinguishable from those of wild-type virus. These F9-deficient virions bound to cells, but their cores did not penetrate into the cytoplasm. Furthermore, cells infected with F9-negative virions did not fuse after a brief low-pH treatment, as did cells infected with virus made in the presence of inducer. In these respects, the phenotype associated with F9 deficiency was identical to that produced by the lack of individual components of a previously described poxvirus entry/fusion complex. Moreover, F9 interacted with proteins of that complex, supporting a related role. Thus, despite the structural relationships of L1 and F9, the two proteins have distinct functions in assembly and entry, respectively.
Methotrexate enhances the antianabolic and antiproliferative effects of 5-aminoimidazole-4-carboxamide riboside
Authors: Beckers Annelies , Organe Sophie , Timmermans Leen , Vanderhoydonc Frank , Deboel Ludo , Derua Rita , Waelkens Etienne , Brusselmans Koen , Verhoeven Guido , Swinnen Johannes V. 
Published: September 2006

Abstract
Because of its ability to mimic a low energy status of the cell, the cell-permeable nucleoside 5-aminoimidazole-4-carboxamide (AICA) riboside was proposed as an antineoplastic agent switching off major energy-consuming processes associated with the malignant phenotype (lipid production, DNA synthesis, cell proliferation, cell migration, etc.). Key to the antineoplastic action of AICA riboside is its conversion to ZMP, an AMP mimetic that at high concentrations activates the AMP-activated protein kinase (AMPK). Here, in an attempt to increase the efficacy of AICA riboside, we pretreated cancer cells with methotrexate, an antimetabolite blocking the metabolism of ZMP. Methotrexate enhanced the AICA riboside–induced accumulation of ZMP and led to a decrease in the levels of ATP, which functions as an intrasteric inhibitor of AMPK. Consequently, methotrexate markedly sensitized AMPK for activation by AICA riboside and potentiated the inhibitory effects of AICA riboside on tumor-associated processes. As cotreatment elicited antiproliferative effects already at concentrations of compounds that were only marginally effective when used alone, our findings on the cooperation between methotrexate and AICA riboside provide new opportunities both for the application of classic antimetabolic chemotherapeutics, such as methotrexate, and for the exploitation of the energy-sensing machinery as a target for cancer intervention. [Mol Cancer Ther 2006;5(9):2211–7]
Targeting changes in cancer: assessing pathway stability by comparing pathway gene expression coherence levels in tumor and normal tissues
Authors: Huang Ruili , Wallqvist Anders , Covell David G. 
Published: September 2006

Abstract
The purpose of this study is to examine gene expression changes occurring in cancer from a pathway perspective by analyzing the level of pathway coherence in tumor tissues in comparison with their normal counterparts. Instability in pathway regulation patterns can be considered either as a result of or as a contributing factor to genetic instability and possibly cancer. Our analysis has identified pathways that show a significant change in their coherence level in tumor tissues, some of which are tumor type specific, indicating novel targets for cancer type–specific therapies. Pathways are found to have a general tendency to lose their gene expression coherence in tumor tissues when compared with normal tissues, especially for signaling pathways. The selective growth advantage of cancer cells over normal cells seems to originate from their preserved control over vital pathways to ensure survival and altered signaling, allowing excessive proliferation. We have additionally investigated the tissue-related instability of pathways, providing valuable clues to the cellular processes underlying the tumorigenesis and/or growth of specific cancer types. Pathways that contain known cancer genes (i.e., “cancer pathways”) show significantly greater instability and are more likely to become incoherent in tumor tissues. Finally, we have proposed strategies to target instability (i.e., pathways that are prone to changes) by identifying compound groups that show selective activity against pathways with a detectable coherence change in cancer. These results can serve as guidelines for selecting novel agents that have the potential to specifically target a particular pathway that has relevance in cancer. [Mol Cancer Ther 2006;5(9):2417–27]
Evidence for the benefits of prophylaxis in the management of hemophilia A
Authors: Nugent Diane , Hoots W. Keith 
Published: September 2006

SummaryThe optimal treatment of hemophilia has been evolving since the advent of factor VIII concentrates, continues to vary geographically, and remains a source of debate. There now exists an extensive clinical literature that demonstrates clear benefits of prophylaxis for patients with hemophilia A compared to on-demand treatment, including a reduction in the number of bleeding episodes, improved joint function, and greater patient well-being. However, the value of these benefits must be weighed against the heavier economic burden of increased factor use. This paper reviews the literature that compares the benefits of prophylaxis with that of on-demand treatment, compares varying prophylaxis dose and administration frequency protocols, and considers the long-term cost-benefit of prophylactic therapy.
Low fasting methionine concentration as a novel risk factor for recurrent venous thrombosis
Authors: Keijzer Miranda , Borm George , Blom Henk , Vollset Stein , Hermus Ad R. M. M. , Ueland Per , den Heijer Martin 
Published: September 2006

SummaryHyperhomocysteinemia is a risk factor for venous thrombosis, but the underlying mechanism is unclear. If the thiol-group of homocysteine interferes with components of the clotting system, we expect that high cysteine will be alsoa risk factor for venous thrombosis. If high homocysteine reflects a disturbed methyl-group donation by S-adenosylmethionine, we expect that low methionine will be a risk factor for thrombosis. We performeda case-control study in 185 patients with recurrent venous thrombosis and in 500 control subjects.We determined methionine, homocysteine, cysteine and assessed the associated thrombotic risk. Low fasting methionine was associated with an increased risk on recurrent venous thrombosis [ORbottom vs. top quartile = 3.3 (95%CI 1.9–5.7)]. Low methionine remained a risk factor [ORbottom vs. top quartile= 3.5 (95%CI 2.0–6.0)] after adjusting for homocysteine and cysteine, whereas the thrombotic risk for homocysteine was lost [OR= 1.0 (95%CI 0.6–1.9)] after adjustment. Cysteine yielded a highest odds ratio of 2.1top vs. bottom quartile (95%CI 1.0–4.0) after adjustment. In conclusion, we found that low fasting methionine is a risk factor for recurrent venous thrombosis.This risk association was stronger for methionine than for homocysteine or cysteine.This supports the hypothesis that impaired methylation may be involved in the pathogenesis of venous thrombosis.
In-vitro and in-vivo consequences of mutations in the von Willebrand factor cleaving protease ADAMTS13 in thrombotic thrombocytopenic purpura
Authors: Donadelli Roberta , Banterla Federica , Galbusera Miriam , Capoferri Cristina , Bucchioni Sara , Gastoldi Sara , Nosari Silvia , Monteferrante Giuseppe , Ruggeri Zaverio , Bresin Elena , Scheiflinger Friedrich , Rossi Edoardo , Martinez Constantino , Coppo Rosanna , Remuzzi Giuseppe , Noris Marina ,   the International Registry of Recurrent and Familial HUS/TTP
Published: September 2006

SummaryThrombotic thrombocytopenic purpura (TTP) is a disease characterized by microvascular thrombosis, often associated with deficiency of the von Willebrand factor (VWF) cleaving protease ADAMTS13.We investigated the spectrum of ADAMTS13 gene mutations in patients with TTP and congenital ADAMTS13 deficiency to establish the consequences on ADAMTS13 processing and activity. We describe five missense (V88M, G1239V, R1060W, R1123C and R1219W), 1 nonsense (W1016Stop) and 1 insertion (82_83insT) mutations. In two patients no mutation was identified despite undetectable protease activity. Expression in HEK293 mammalian cells (V88M, G1239V, R1123C and R1219W) documented that three missense mutants were not secreted, whereas theV88M was secreted at low levels and with reduced activity. We also provide evidence that impaired secretion of ADAMTS13 mutants observed in vitro translates into severely reduced ADAMTS13 antigen levels in patients in vivo. To evaluate whether the small amounts of mutant protease present in the circulation of patients had VWF cleaving activity, WT and mutant rADAMTS13 were stably expressed in Drosophila S2 cells under the influence of the Drosophila BiP protein signal sequence, which allows protein secretion. Drosophila expression system showed a 40–60% protease activity in the mutants. Several single nucleotide polymorphisms (SNPs) within exons and intron boundaries were found in patients, suggesting that the interplay of SNPs could at least in part account for ADAMTS13 functional abnormalities in patients without mutations. In conclusion, defective secretion and impaired activity of the mutants concur to determine an almost complete deficiency of ADAMTS13 activity in patients with a homozygous or two heterozygous ADAMTS13 mutations.
Plasma glycoprotein V levels in the general population: Normal distribution, associated parameters and implications for clinical studies
Authors: Meyer Nicolas , Wolff Valérie , Kientz Daniel , Wiesel Marie-Louise , Gachet Christian , Cazenave Jean-Pierre , Aleil Boris , Lanza François 
Published: September 2006

SummarySoluble glycoproteinV (sGPV) isa new plasma marker of thrombosis released from the platelet surface by thrombin. sGPV levels are increased in patients with atherothrombotic diseases, but the determinants of sGPV levels are unknown in the general population. Identification of these potential confounding factors is needed for correct design and analysis of clinical studies on cardiovascular diseases.The aim of this study was to determine the normal range of plasma values and the factors controlling sGPV levels in a population of normal individuals.Three hundred blood donors were recruited at the Etablissement Français du Sang-Alsace for the measurement of plasma levels of sGPV, platelet factor 4 (PF4), thrombin-antithrombin complexes (TAT) and D-dimers.The plasma level of sGPV was (median [interquartile range]) 27.5 [23.5–34.4] µg/l and displayed a Gaussian distribution. sGPV had a lower interindividual coefficient of variation (33%) than PF4 (176%), TAT (87%) or D-dimers (82%). sGPV levels were independent of age and sex but sensitive to red cell (r=0.412; p<0.0001) and platelet counts (r=0.267; p=0.001), total cholesterol (r=−0.313; p<0.0001), food intake (r=0.184; p=0.0014) and smoking (r=−0.154; p=0.039). Contrary to PF4 and TAT, sGPV did not differ between venous and arterial blood samples of 12 healthy individuals. Red cell and platelet counts, total cholesterol, current smoking and recent food intake are important determinants of sGPV levels and must be taken into account in clinical studies using sGPV as a thrombosis marker. Normal distribution of sGPV levels in the general population supports its use in clinical applications.
Gene polymorphisms implicated in influencing susceptibility to venous and arterial thromboembolism: Frequency distribution in a healthy German population
Authors: Tolou Farzaneh , Dörner Thomas , Kiesewetter Holger , Salama Abdulgabar , Hoppe Berthold 
Published: September 2006

SummaryEvolvement and progression of cardiovascular diseases affecting the venous and arterial system are influenced bya multitude of environmental and hereditary factors. Many of these hereditary factors consist of defined gene polymorphisms, such as single nucleotide polymorphisms (SNPs) or insertion-deletion polymorphisms, which directly or indirectly affect the hemostatic system. The frequencies of individual hemostatic gene polymorphisms in different normal populations are well defined. However, descriptions of patterns of genetic variability of a larger extent of different factors of hereditary hypercoagulability in single populations are scarce. The aim of this study was i) to givea detailed description of the frequencies of factors of hereditary thrombophilia and their combinations in a German population (n = 282) and ii) to compare their distributions with those reported for other regions. Variants of coagulation factors [factor V 1691G>A (factor V Leiden), factor V 4070A>G (factor V HR2 haplotype), factor VII Arg353Gln, factor XIII Val34Leu, β-fibrinogen –455G>A, prothrombin 20210G>A], coagulation inhibitors [tissue factor pathway inhibitor 536C>T, thrombomodulin 127G>A], fibrinolytic factors [angiotensin converting enzyme intron 16 insertion/deletion, factor VII-activating protease 1601G>A (FSAP Marburg I), plasminogen activator inhibitor 1–675 insertion/deletion (5G/4G), tissue plasminogen activator intron h deletion/insertion], and other factors implicated in influencing susceptibility to thromboembolic diseases [apolipoprotein E2/E3/E4, glycoprotein Ia 807C>T, methylenetetrahydrofolate reductase 677C>T] were included. The distribution of glycoprotein Ia 807C>T deviated significantly from the Hardy- Weinberg equilibrium, and a comparison with previously published data indicates marked region and ethnicity dependent differences in the genotype distributions of some other factors.